Antiviral agents and methods of treating viral infections

ABSTRACT

The present invention relates to methods of treating viral or fungal infections using 3-aminopyridine-2-carboxyaldehyde thiosemicarbazone (3-AP) and 3-amino-4-methylpyridine-2-carboxaldehyde thiosemicarbazone (3-AMP) and its prodrug forms and to pharmaceutical compositions comprising these compounds.

RELATED APPLICATIONS

This application claims the benefit of provisional paten application60/285,559 filed Apr. 20, 2001.

FIELD OF THE INVENTION

The present invention relates to methods of treating viral infectionsusing 3-aminopyridine-2-carboxyaldehyde thiosemicarbazone (3-AP,Triapine™) and 3-amino-4-methylpyridine-2-carboxaldehydethiosemicarbazone (3-AMP) and their prodrug forms and to pharmaceuticalcompositions comprising these compounds. Combination therapy with otherantiviral agents, in particular, nucleoside antiviral agents, representsanother aspect of the present invention.

BACKGROUND

Triapine is a ribonucleotide reductase (RNR) inhibitor that reduces thecellular pool of DNA precursors (dNTPs) by interfering with their denovo synthesis (Cory et al., (1994) Biochem. Pharmacol. 48, 335-44).Depletion of dNTPs resulted in inhibition of DNA synthesis. Triapine wasfirst developed as an anti-cancer agent for its action against thegrowth of tumor cells both in vitro and in vivo (Liu et al., (1992) J.Med. Chem. 35, 3672-77). It has recently been shown that concentrationof Triapine reached 1.0 micromolar in cancer patients receiving a96-hour infusion of Triapine at a dose of 96 mg/mm2 (Modiano et al.,Proc. Am. Assoc. Cancer Res., 42, 834, 2001). The mechanism of action ofTriapine is similar to that of another anti-cancer agent, hydroxyurea,which has been approved for treatment of cancers in humans.

Recently, hydroxyurea, another known RNR inhibitor, was shown to havesynergistic effects against HIV-infected cells (human immunodeficiencyviruses, the causative agents of AIDS) when combined with2′,3′-dideoxyinosine (DDI) (Gao et al., (1998) Biochem. Pharmacol. 56,105-12). The mechanism of action is unknown, but may be due to thedepletion of dNTPs in cells treated with hydroxyurea.

3-AP and 3-AMP, like other thiosemicarbazone analogs of this class(Springarn and Sartorelli, J. Med. Chem. (1979) 22, 1314-6), have verystrong iron binding affinity and are capable of removing iron fromferritin. Iron is required RNR activity and for normal physiologicalfunction of organisms and iron deprivation inhibits proliferation ofprotozoa (Merali et al., Antimicrob. Agents Chemother. (1996) 40,1298-1300), bacteria (Lowy et al., Antimicrob. Agents Chemother. (1984)25, 375-6), fungi (Newman et al., Antimicrob. Agents Chemother. (1995)39, 1824-9; Shulman et al., Arzneimittelforschung (1972) 22, 154-8;Kerbs et al., Sabouraudia (1979), 17, 241-50), and viruses (Dai et al.,Virology (1994) 205, 210-6; Cinatl et al., Antiviral Res. (1994) 25,73-77; Bayraktar et al., J. Viral Hepat. (1996) 3, 129-35; Martelius etal, Transplantation (1999) 15, 1753-61; Georgiou et al, J. Infect. Dis.(2000) 181, 484-90). In addition to depletion of intracellular dNTPpools, that 3-AP inhibits viral dissemination could be mediated throughits iron chelating properties (Chouteau et al., J. Hepatol. (2001) 34,108-13; Georgiou et al, J. Infect. Dis. (2000) 181, 484-90; Bayraktar etal., J. Viral Hepat. (1996) 3, 129-35; Conti et al., Boll. Ist. SieroterMilan (1990) 69, 431-6).

3-AP, because of its strong iron-chelating property, can be used toremove excessive tissue iron in sickle cell disease patients who requireregular blood transfusion (Cohen and Martin, Semin. Hematol. (2001) 38(Suppl. 1), 69-72). As a potent inhibitor of RNR, 3-AP could also beused for the treatment of psoriasis (Smith, Clin. Exp. Dermatol. (1999)24, 2-6).

Chronic HBV (hepatitis B virus) infection remains a therapeuticchallenge for clinicians. The recent development of lamivudine hasprovided new hope in the therapy of chronic hepatitis B. However, due tothe slow kinetics of viral clearance and the spontaneous geneticvariability of HBV, lamivudine therapy is associated with the selectionof drug resistant mutants in up to 50% of patients after 3 years oftherapy. It is therefore important to continue research to develop newanti-HBV strategies using in vitro and in vivo evaluation inexperimental models of HBV replication.

Herpes simplex virus (HSV) encodes a RNR which is similar to the oneencoded by mammalian cells. HSV replication does not require theexpression of viral RNR in exponentially growing cells but is requiredfor viral replication in quiescent cells (Goldstein and Weller, (1988)Virol. 166, 1-51). Duan et al., Antimicrob. Agents Chemother. (1998) 42,1629-35, showed that the RNR inhibitor BILD1633 SE in combination withacyclovir had activity against acyclovir-resistant HSV strains.

Recently, flavivirus infections, including West Nile virus infections,have become increasingly frequent in the United States. The flavivirusesare agents of infectious disease which predominate in East, Southeastand South Asia and Africa, although they may be found in other parts ofthe world as well. Japanese encephalitis virus is the causative agent ofJapanese encephalitis (JE). The mortality rate from JE is rather highand the disease brings heavy sequelae. Although found in Japan, thedisease has spread to other parts of Asia and is now found predominantlyoutside of Japan, primarily in South and Southeast Asia.

Dengue viruses are causative agents of dengue fever/dengue hemorrhagicfever. Infection with dengue viruses is a major public health problem intropical countries, expecially in Southeast Asia and the WesternPacific, but dengue viruses may also be found in the Americas. As thedengue virus is transmitted to humans via the Aedes aegypti mosquito, itis not unexpected that tropical and subtropical countries, inparticular, those in Southeast Asia, are highly endemic for dengue.

Viral replication requires dNTPs, and depletion of intracellular dNTPsby Triapine may prevent viruses from multiplying. In addition, this newstrategy may be used in combination with other anti-viral agents totreat, or to prevent or delay the development of drug resistant mutants.

OBJECTS OF THE INVENTION

It is an object of the invention to provide novel combinationpharmaceutical compositions for treating viral infections in patients.

It is another object of the invention to provide novel methods forreducing viral growth, elaboration and replication and for treatingviral infections in patients.

It is also an object of the invention to provide methods for treatingfungal infections in patients.

Any one or more of these and/or other objects of the present inventionmay be readily gleaned from a review of the description of the presentinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a representation of certain chemical embodiments according tothe present invention.

FIGS. 2-3 are representations of chemical schemes for synthesizingcompounds according to the present invention.

FIG. 4 depicts the antiviral activity of combinations of Triapine withD4T, ddI and AZT.

SUMMARY OF THE INVENTION

The present invention relates to methods for inhibiting the growth,replication or elaboration of a virus population or for treating avariety of virus infections, including, for example, humanimmunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), human T-cellleukemia viruses 1 and 2 (HTLV-1 and HTLV-2), respiratory syncytialvirus (RSV), human papilloma virus (HPV), adenovirus, hepatitis B virus(HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), varicellazoster virus (VZV), cytomegalovirus (CMV), herpes simplex viruses 1 and2 (HSV-1 and HSV-2), human herpes virus 8 (HHV-8, also known as Kaposi'ssarcoma-associated virus) and flaviviruses, including Yellow Fevervirus, Dengue virus, Japanese Encephalitis and West Nile viruses, saidmethod comprising administering an anti-viral effective amount of acomposition according to the present invention to a patient in needthereof to treat, prevent or reduce the likelihood of contracting aviral infection.

The present invention therefore relates to a method of treatinginhibiting the growth, replication and/or the elaboration of a viralpopulation or a viral infection in a patient, comprising administrationto said patient an effective amount of a compound according to thestructure:

Where R¹ is H or a C₁-C₃ alkyl group, preferably H or CH₃;R² is H or CO₂R³;R³ is CHRR′ or

where R is H, CH₃, CH₂CH₃, CH₂CH₂CH₃, i-propyl;R′ is a free acid phosphate, phosphate salt or S—S—R″ group;R″ is CH₂CH₂NHR⁴, CH₂CH₂OH, CH₂COOR⁵, ortho- or para-substituted C₁-C₃alkylphenyl or ortho or para nitrophenyl;R⁴ is H or a C₁-C₁₈ acyl group (preferably, a C₁-C₄ group), benzoyl, ora substituted benzoyl group;R⁵ is H, a C₁-C₁₈ alkyl group (preferably, a C₁-C₃ group), phenyl,substituted phenyl, benzyl, or a substituted benzyl group;R⁶, R⁷, R⁸, R⁹ and R¹⁰ are independently selected from H, a free acidphosphate, a phosphate salt, or an S—S—R″ group, a C₁-C₃ alkyl group, F,Cl, Br, I, OCH₃, OCF₃, CF₃, NO₂, CN, SO₂CF₃, SO₂CH₃, COOCH₃, SF₅, COCH₃,NH₂, N(CH₃)₂, SCH₃ or OH;With the proviso that when any two of R⁶, R⁷, R⁸, R⁹ and R¹⁰ are otherthan H, the other of R⁶, R⁷, R⁸, R⁹ and R¹⁰ are H, and with the provisothat no more than one of R⁶, R⁷, R⁸, R⁹ and R¹⁰ is a free acidphosphate, a phosphate salt or S—S—R″.

In preferred aspects of the present invention, R⁶, R⁸ or R¹⁰ is a freeacid phosphate or a phosphate salt. In other preferred aspects of thepresent invention, R⁶ is a free acid phosphate or phosphate salt and theother of R⁷, R⁸, R⁹ and R¹⁰ are H. In still other aspects of the presentinvention R¹ and R² are both H.

The invention provides methods of use relating to treatment ofinfections caused by viruses, including, for example, humanimmunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), human T-cellleukemia viruses 1 and 2 (HTLV-1 and HTLV-2), respiratory syncytialvirus (RSV), human papilloma virus (HPV), adenovirus, hepatitis B virus(HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), varicellazoster virus (VZV), cytomegalovirus (CMV), herpes simplex viruses 1 and2 (HSV-1 and HSV-2), human herpes virus 8 (HHV-8, also known as Kaposi'ssarcoma-associated virus) and flaviviruses, including Yellow Fevervirus, Dengue virus, Japanese Encephalitis and West Nile viruses. Themethod includes the use of prodrug forms of 3-AP and 3-AMP as otherwisedescribed herein for the treatment of viral infections.

In alternative embodiments, compounds according to the present inventionmay be used to treat fungal infections including, for example,infections caused by Piedraia hortae, Trichosporon beigelii, Malasseziafurfur, Epidermophyton spp., Microsporum spp., Trichophyton spp.,Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans,Histoplasma capsulatum, Aspergillus spp. and Candida albicans. Methodsof treating fungal infections comprising administering an anti-fungaleffective amount of one or more of 3-AP, 3-AMP or one of its prodrugs asotherwise described herein.

Compositions according to the present invention may be used alone or incombination with reverse transcriptase inhibitors, protease inhibitors,other immunomodulators, all for the treatment of HIV infections, as wellas other viral infections. The invention also provides methods of usefor the treatment of HBV infections by combining one or more compoundsaccording to the present invention along with reverse trancriptaseinhibitors, or interferon, or both. The invention also provides a methodfor the treatment of HSV infection by combining an effective amount ofone or more of the compositions as described above with one or moreanti-HSV agents such as acyclovir, DDI and D4T in amounts effective totreat the viral infection. Without being limited by way of theory, it isbelieved that the use of the presently described compounds exhibit theiranti-viral activity primarily through inhibition of cellular and viralRNR and/or chelation of divalent metal ions such as iron, among others.Use of compositions in combination therapy which inhibit viruses in amanner other than through RNR are particularly preferred as they oftenprovide synergistic anti-viral activity in combination with 3-AP (R¹ andR² are H), 3-AMP (R¹ is CH₃ and R² is H) and prodrug compositionsaccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The following terms shall be used throughout the specification todescribe the present invention.

The term “patient” is used throughout the specification to describe ananimal, preferably a human, to whom treatment, including prophylactictreatment, with the compositions according to the present invention isprovided. For treatment of those infections, conditions or diseasestates which are specific for a specific animal such as a human patient,the term patient refers to that specific animal.

The term “virus” shall be used to describe all types of viruses, thegrowth or replication of which may be inhibited or disease states ofwhich may be treated using one or more methods according to the presentinvention. Viruses which may be treated according to the presentinvention include, for example, human immunodeficiency viruses 1 and 2(HIV-1 and HIV-2), human T-cell leukemia viruses 1 and 2 (HTLV-1 andHTLV-2), respiratory syncytial virus (RSV), human papilloma virus (HPV),adenovirus, hepatitis B virus (HBV), hepatitis C virus (HCV),Epstein-Barr virus (EBV), varicella zoster virus (VZV), cytomegalovirus(CMV), herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), human herpesvirus 8 (HHV-8, also known as Kaposi's sarcoma-associated virus) andflaviviruses, including Yellow Fever virus, Dengue virus, JapaneseEncephalitis and West Nile viruses, among numerous others.

The term “human immunodeficiency virus” shall be used to describe humanimmunodeficiency virus (HIV) and its infections, which term shall beused to embrace both human immunodeficieny virus 1 (HIV-1) and humanimmunodeficiency virus 2 (HIV-2).

The term “human T-cell leukemia virus” shall be used to describe humanT-cell leukemia virus and its infections, which term shall be used toembrace both human T-cell leukemia virus 1 (HTLV-1) and human T-cellleukemia virus 2 (HTLV-2).

The term “Hepatitis B Virus” (HBV) is used to describe the virus (serumhepatitis virus) which produces viral heptatis type B in humans. This isa viral disease with a long incubation period (about 50 to 160 days) incontrast to Hepatitis A virus (infectious hepatitis virus) which has ashort incubation period. The virus is usually tramsmitted by injectionof infected blood or blood derivatives or merely by use of contaminatedneedles, lancets or other instruments. Clinically and pathologically,the disease is similar to viral hepatitis type A; however, there is nocross-protective immunity. Viral antigen (HBAg) is found in the serumafter infection.

The term “Hepatitis C Virus” or (HCV) is used throughout thespecification to describe the hepatitis virus which is the causativeagent of non-A, non-B hepatitis. The disease in the acute stage is, ingeneral, milder than hepatitis B, but a greater proportion of suchinfections become chronic.

The term “Epstein-Barr virus” (EBV) is used throughout the specificationto describe a herpetovirus found in cell cultures of Burkitt's lymphoma.EBV is the causative agent in infectious mononucleosis, as well as in anumber of other related conditions/disease states, includingEBV-associated lymphomas.

The term “Varicella-Zoster virus” (VZV) is used to describe Herpesvirusvaricellae, also known as chicken pox or herpes zoster. Varicellaresults from a primary infection with the virus; herpes zoster resultsfrom secondary invasion by the same or by reactivation of infectionwhich in many instances may have been latent for a number of years. Boththe primary and secondary infections of VZV may be treated usingcompositions according to the present invention.

The term “respiratory syncytial virus” (RSV) is used throughout thespecification to describe an RNA-containing virus of the genusPneumovirus that causes minor respiratory infection with rhinitis andcough in adults, but is capable of causing bronchitis andbronchopneumonia in young children. The virus is named for the tendencyto form syncytia in tissue culture.

The term “adenovirus” is used throughout the specification to describe avirus of the family adenoviridae which are doublestranded DNA-containingviruses, which infect mammals and birds. The virion is 70 to 90 nm indiameter and is naked (has no envelope). The virus develops in nuclei ofinfected cells; isolation requires tissue cultures since laboratoryanimals are not susceptible to apparent infection. The family includestwo genera, Mastadenovirus and Acviadenovirus.

The term “Human Herpes Virus 8” (HHV-8) is used throughout thespecification to describe a herpetovirus which is believed to be thecausative agent of Kaposi's sarcoma in AIDS patients.

The term “Human Papilloma Virus” (HPV) is used throughout thespecification to describe a virus which causes genital warts. Also knownas infectious warts virus, HPV is a universal, common, often recurrentviral infection with a large number of serotypes. HPV infection can leadto the formation of genital warts which can, in turn, lead to genitaland/or cervical cancer. Genital warts caused by HPV types 1, 2, 6, 11,16 and 18 are generally transmitted sexually and are often associatedwith cervical and/or genital cancer. HPV may mature to produce apapillary tumor or wart, which is a circumscribed benign epithelialtumor projecting from the surrounding surface. It is generally a benignepithelial neoplasm consisting of villous or arborescent outgrowths offibrovascular stroma covered by neoplastic cells.

The term “flavivirus” is used throughout the specification to describeviruses belonging to the genus Flavivirus of the family Togaviridae.According to virus taxonomy, about 50 viruses including Hepatitis Cvirus (HCV), Yellow Fever virus, Dengue Virus, Japanese Encephalitisvirus, West Nile virus and related flaviviruses are members of thisgenus. The viruses belonging to the genus Flavivirus are simply calledflaviviruses. These viruses were formerly classified as group Barboviruses. The flaviviruses are agents of infectious disease andpredominate in East, Southeast and South Asia and Africa, although theymay be found in other parts of the world as well.

The term “Yellow Fever virus” is used to describe the flavivirus whichis the causative agent of yellow fever. Yellow fever is a tropicalmosquito-borne viral hepatitis, due to Yellow Fever virus (YFV), with anurban form transmitted by Aedes aegypti, and a rural, jungle or sylvaticform from tree-dwelling mammals by various mosquitos of the Haemagogusspecies complex. Yellow fever is characterized clinically by fever, slowpulse, albuminuria, jaundice, congesion of the face and hemorrhages,especially hematemesis (“black vomit”). It is fatal in about 5-10% ofthe cases.

The term “Dengue virus” is used throughout the specification to descibethe flavivirus which is the causative agent(s) of dengue fever/denguehemorrhagic fever. Dengue is a disease of tropical and subtropicalregions occurring epidemically and caused by Dengue virus, one of agroup of arboviruses which causes the hemorrhagic fever syndrome. Fourgrades of severity are recognized: grade I: fever and constitutionalsymptoms, grade II: grade I plus spontaneous bleeding (of skin, gums orgastrointestinal tract), grade III: grade II plus agitation andcirculatory failure and grade IV: profound shock. The disease istransmitted by a mosquito of the genus Aedes (generally A. aegyptiI, butfrequently, A. albopictus). Also called Aden, bouquet, breakbone, dandy,date, dengue (hemorrhagic) or polka, solar fever, stiffneck fever,scarlatina rheumatica or exanthesis arthorosia. “Hemorrhagic dengue” isa more pathogenic epidemic form of dengue which has erupted in a numberof epidemic outbreaks in the Pacific region in recent years.

The term ‘West Nile virus” is used to describe the flavivirus which isthe causative agent of West Nile fever, a disease characterized byheadache, fever, masculopapular rash, myalgia, lymphadenopathy andleukopenia. The virus is spread by Culex mosquitoes from a reservoir inbirds. Although in the past, West Nile virus infections had beenconsidered virtually nonexistent in the United States, recentdevelopments have suggested that West Nile and other flavivirusinfections will appear with greater regulatory in the future in theUnited States.

The term fungus shall mean “fungus” as that term is generally known inthe art. Fungal infections which may be treated using 3-AP, 3-AMP andtheir prodrug forms alone or in combination with other anti-fungalagents including, for example infections caused by Piedraia hortae,Trichosporon beigelii, Malassezia furfur, Epidermophyton spp.,Microsporum spp., Trichophyton spp., Blastomyces dermatitidis,Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum,Aspergillus spp., Candida albicans.

The term “anti-fungal agent” shall used to describe a compound which maybe used to treat a fungus infection other than 3-AP, 3-AMP or prodrugsof 3-AP and 3-AMP according to the present invention. Anti-fungal agentsaccording to the present invention include, for example, terbinafine,fluconazole, itraconazole, posaconazole, clotrimazole, griseofulvin,nystatin, tolnaftate, caspofungin, amphotericin B, liposomalamphotericin B, and amphotericin B lipid complex.

The term “pharmaceutically acceptable salt” is used throughout thespecification to describe a salt form of one or more of the compositions(and in particularly preferred aspects according to the presentinvention, phosphate salts) herein which are presented to increase thesolubility of the compound in saline for parenteral delivery or in thegastric juices of the patient's gastrointestinal tract in order topromote dissolution and the bioavailability of the compounds.Pharmaceutically acceptable salts include those derived frompharmaceutically acceptable inorganic or organic bases and acids.Suitable salts include those derived from alkali metals such aspotassium and sodium, alkaline earth metals such as calcium, magnesiumand ammonium salts, among numerous other acids well known in thepharmaceutical art. Sodium and potassium salts are particularlypreferred as neutralization salts of carboxylic acids and free acidphosphate containing compositions according to the present invention.

The term “inhibitory effective concentration” or “inhibitory effectiveamount” is used throughout the specification to describe concentrationsor amounts of compounds according to the present invention whichsubstantially or significantly inhibit the growth or replication ofsusceptible viruses, especially including human immunodeficiency viruses1 and 2 (HIV-1 and HIV-2), human T-cell leukemia viruses 1 and 2 (HTLV-1and HTLV-2), respiratory syncytial virus (RSV), human papilloma virus(HPV), adenovirus, hepatitis B virus (HBV), hepatitis C virus (HCV),Epstein-Barr virus (EBV), varicella zoster virus (VZV), cytomegalovirus(CMV), herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), human herpesvirus 8 (HHV-8, also known as Kaposi's sarcoma-associated virus) andflaviviruses, including Yellow Fever virus, Dengue virus, JapaneseEncephalitis and West Nile viruses, among numerous others. This termalso refers to amounts of concentrations of compounds (whether 3-AP,3-AMP or its prodrugs or more traditional anti-fungal agents asdescribed in the present specification) which inhibit the growth offungi.

The term “therapeutic effective amount” or “therapeutically effectiveamount” is used throughout the specification to describe concentrationsor amounts of compounds according to the present invention which aretherapeutically effective in treating viruses or fungi according to thepresent invention.

The term “preventing effective amount” is used throughout thespecification to describe concentrations or amounts of compoundsaccording to the present invention which are prophylactically effectivein preventing, reducing the likelihood of infection or delaying theonset of infections in patients caused by human immunodeficiency viruses1 and 2 (HIV-1 and HIV-2), human T-cell leukemia viruses 1 and 2 (HTLV-1and HTLV-2), respiratory syncytial virus (RSV), human papilloma virus(HPV), adenovirus, hepatitis B virus (HBV), hepatitis C virus (HCV),Epstein-Barr virus (EBV), varicella zoster virus (VZV), cytomegalovirus(CMV), herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), human herpesvirus 8 (HHV-8, also known as Kaposi's sarcoma-associated virus) andflaviviruses, including Yellow Fever virus, Dengue virus, JapaneseEncephalitis and West Nile viruses, among numerous others. This termshall also be used to describe amounts or concentrations of anti-fungalagents which are prophylactically effective in preventing, reducing thelikelihood or delaying the onset of a fungal infection.

The term “effective amount” shall mean an amount or concentration of acompound according to the present invention which is effective withinthe context of its administration or use, including, for example, thetreatment or prevention of viral and/or fungal infections.

The term “coadministration” or “combination therapy” is used to describea therapy in which at least two active compounds in effective amountsare used to treat a viral or fungal infection at the same time. Althoughthe term coadministration preferably includes the administration of twoactive compounds to the patient at the same time, it is not necessarythat the compounds be administered to the patient at the same time,although effective amounts of the individual compounds will be presentin the patient at the same time.

The term “alkyl” is used throughout the specification to describe ahydrocarbon radical containing between one and three carbon units. Alkylgroups for use in the present invention include linear or branched-chaingroups, such as propyl and isopropyl.

The present invention relates to the use of 3-AP and 3-AMP, includingtheir prodrug forms, as otherwise described herein for the inhibition ofviral infections and the treatment of viral diseases in animals,including humans. Viruses which can be treated by the present inventioninclude, for example, human immunodeficiency viruses 1 and 2 (HIV-1 andHIV-2), human T-cell leukemia viruses 1 and 2 (HTLV-1 and HTLV-2),respiratory syncytial virus (RSV), human papilloma virus (HPV),adenovirus, hepatitis B virus (HBV), hepatitis C virus (HCV),Epstein-Barr virus (EBV), varicella zoster virus (VZV), cytomegalovirus(CMV), herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), human herpesvirus 8 (HHV-8, also known as Kaposi's sarcoma-associated virus) andflaviviruses, including Yellow Fever virus, Dengue virus, JapaneseEncephalitis and West Nile viruses, among numerous others.

The present invention also describes the use of 3-AP and 3-AMP and itsprodrug forms in combination with other agents which may be used totreat viral infections and to prevent a viral infection or reduce thelikelihood that an exposure of an animal to a virus will result in aviral infection in that animal. Another aspect of the present inventionrelates to combination therapy with at least one compound according tothe present invention, in combination with at least one additionalanti-viral agent which inhibits viruses by a mechanism other than byinhibition of viral RNR, which combination therapy produces synergisticinhibition of viral infections.

The present invention may be used in the treatment of HIV infections.For example, compositions according to the present invention may becombined with other anti-HIV agents, especially including, for example,D4T, DDI, AZT, lamivudine,Beta-L-5-Fluoro-2′,3′-dideoxydidehydrocytidine (β-LFd4C),Beta-L-5-Fluoro-2′,3′-dideoxycytidine (β-LFddC),Beta-L-2′,3′-dideoxydidehydrocytidine (β-Ld4C) or other nucleosideanti-HIV agents for the treatment of AIDS related conditions such asAIDS-related complex (ARC) and AIDS-related neurological conditions. Thepresent invention is also useful in the prevention or the reduction ofthe likelihood of progression to clinical illness of individuals who areanti-HIV antibody or HIV-antigen positive and in prophylaxis followingexposure to HIV.

The present invention may also be used in the treatment of HBVinfections, including the use in combination with other anti-HBV agentsfor the treatment of acute or chronic HBV infections, especiallyincluding for example, lamivudine,Beta-L-5-Fluoro-2′,3′-dideoxydidehydrocytidine (β-LFd4C),Beta-L-5-Fluoro-2′,3′-dideoxycytidine (β-LFddC) or other nucleosideanti-HBV agents. The invention is also useful in the prevention or thereduction of the likelihood of progression to clinical illness ofindividuals who are anti-HBV antibody or HBV-antigen positive and inprophylaxis following potential exposure to HBV such as after a livertransplant.

The present invention is also directed to the use of one or morecompounds as otherwise described herein in combination with otheranti-HSV agents for the treatment of HSV infections, especially forexample, acyclovir (ACV). The invention is also useful in the preventionor the reduction of the likelihood of progression to clinical illness ofindividuals who are infected with HSV or in prophylaxis followingpotential exposure to HSV.

The present invention also relates to compositions and methods for usein treating fungal infections either alone or using combinations ofagents. Suitable therapeutic agents for use in such combination include3-AP, 3-AMP or one or more of its prodrug forms as described in detailhereinabove, alone or in combination with another anti-fungal agentssuch as terbinafine, fluconazole, itraconazole, posaconazole,clotrimazole, griseofulvin, nystatin, tolnaftate, caspofungin,amphotericin B, liposomal amphotericin B, and amphotericin B lipidcomplex.

In another aspect, the present invention is directed to the use of oneor more compounds according to the present invention in apharmaceutically acceptable carrier in combination with at least oneother anti-viral agent at a suitable dose ranging from about 1 to about100 mg/kg of body weight per day, preferably within the range of about 2to 50 mg/kg/day, most preferably in the range of 3 to 20 mg/kg/day. Thedesired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example as two,three, four or more sub-doses per day.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.05 to about5 uM, preferably about 0.1 to 2 uM, most preferably about 0.2 to about 1uM. This may be achieved, for example, by the intravenous injection ofabout a 0.1 to 10% solution of the active ingredient, optionally insaline, or orally administered as a bolus containing about 0.1 to about5 g of the active ingredient. Desirable blood levels may be maintainedby a continuous infusion to preferably provide about 0.01 to about 2.0mg/kg/hour or by intermittent infusions containing about 0.4 to about 15mg/kg of the active ingredient. Oral dosages, where applicable, willdepend on the pharmacokinetics of the compounds to be administered.While it is possible that, for use in therapy, a compound of theinvention may be administered as the raw chemical it is preferable topresent the active ingredient as a pharmaceutical formulation.

Pharmaceutical formulations include those suitable for oral, rectal,nasal, topical (including buccal and sub-lingual), vaginal or parenteral(including intramuscular, sub-cutaneous and intravenous) administration.Compositions according to the present invention may also be presented asa bolus, electruary or paste. Tablets and capsules for oraladministration may contain conventional excipients such as bindingagents, fillers, lubricants, disintegrants, or wetting agents. Thetablets may be coated according to methods well known in the art. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may contain conventionaladditives such as suspending agents, emulsifying agents, non-aqueousvehicles (which may include edible oils), or preservatives.

When desired, the above described formulations may be adapted to providesustained release characteristics of the active ingredient(s) in thecomposition.

In the pharmaceutical aspect according to the present invention, thecompound(s) according to the present invention is formulated preferablyin admixture with a pharmaceutically acceptable carrier. In general, itis preferable to administer the pharmaceutical composition parenterallyand in particular, in intravenously or intramuscular dosage form, but anumber of formulations may be administered via other parenteral routes,such as transdermal, buccal, subcutaneous, suppository or other route,including via an oral route of administration. Intravenous andintramuscular formulations are preferably administered in sterilesaline. Of course, one of ordinary skill in the art may modify theformulations within the teachings of the specification to providenumerous formulations for a particular route of administration withoutrendering the compositions of the present invention unstable orcompromising their therapeutic activity. In particular, the modificationof the present compounds to render them more soluble in water or othervehicle, for example, may be easily accomplished by minor modifications(such as salt formulation, etc.) which are well within the ordinaryskill in the art. It is also well within the routineer's skill to modifythe route of administration and dosage regimen of a particular compoundin order to manage the pharmacokinetics of the present compounds formaximum beneficial effect to the patient.

The routineer will take advantage of favorable pharmacokineticparameters of the pro-drug forms of the present invention, whereapplicable, in delivering the present compounds to a patient sufferingfrom a viral infection to maximize the intended effect of the compound.

The pharmaceutical compositions according to the invention may alsocontain other active ingredients such as antimicrobial agents,antiinfective agents, or preservatives.

The invention thus provides, in a further aspect, a pharmaceuticalcomposition combination comprising an effective amount of 3-AP, 3-AMP orone or more of its prodrug forms as otherwise described herein or apharmaceutically acceptable derivative thereof together with anothertherapeutically active agent, and in particular, another antiviralagent. Effective amounts or concentrations of each of the activecompounds are to be included within the pharmaceutical compositionsaccording to the present invention.

Pharmaceutical formulations comprising at least one of 3-AP, 3-AMP andprodrug forms presented in combination with an effective amount of atleast one additional anti-viral agent in further combination with apharmaceutically acceptable carrier, represent a further aspect of thepresent invention.

The present invention also relates to compositions and methods for usein treating viral infections using combinations of agents. Suitabletherapeutic agents for use in such combinations with 3-AP, 3-AMP or oneor more of its prodrug forms include acyclic nucleosides such asacyclovir or ganciclovir, interferons such as alpha., beta orgamma.-interferon, reverse transcriptase inhibitors and nucleosides,transport inhibitors such as dipyridamole, 2′,3′-dideoxynucleosides(including β-L-FddC), 2,3′-dideoxy-2′,3′-didehydronucleosides (includingβ-L-Fd4C), 3TC (lamivudine), AZT, 2′,3′-dideoxycytidine (DDC),2′,3′-dideoxyadenosine, 2′,3′-dideoxyinosine (DDI),2′,3′-dideoxythymidine (DDT), 2′,3′-dideoxy-2′,3′-didehydrothymidine(D4T) and 2′,3′-dideoxy-2′,3′-didehydrocytidine (D4C), tenofirir DF,adefovir, dipivoxil, immunomodulators such as interleukin II (IL2) andgranulocyte macrophage-colony stimulating factor (GM-CSF),erythropoetin, ampligen, thymodulin, thymopentin, foscarnet, ribavirinand inhibitors of HIV binding to CD4 receptors e.g. soluble CD4, CD4fragments, CD4 hybrid molecules, glycosylation inhibitors such as2-deoxy-D-glucose, castanospermine and 1-deoxynojirimycin.

The individual components of such combinations maybe administered eithersequentially or simultaneously in separate or combined pharmaceuticalformulations.

When one or more of the compounds according to the present invention isused in combination with a second therapeutic agent active against thesame virus or fungus the dose of each compound may be either the same asor differ from that when the compound is used alone. Appropriate doseswill be readily appreciated by those skilled in the art.

Chemical Synthesis

Compositions according to the present invention are synthesized usingthe general and synthetic methods which are set forth in U.S. Pat. No.5,767,134, issued Jun. 16, 1998 and as otherwise set forth herein.Additional prodrug forms of 3-AP and 3-AMP are synthesized by themethods which may be followed in the '134 patent as well as methodswhich are described below.

A number of phosphate bearing prodrugs (as set forth in FIG. 1) weresynthesized readily in good quantities and evaluated. The disodium saltsof these prodrugs were very soluble in water.

As set forth in attached FIG. 2, the 5-chloro prodrug compound 6 wassynthesized as shown. Thus, the acid 20 was prepared from, for example,2-chloro-3-nicotinic acid methyl ester 18 or a related derivative in atwo-step sequence consisting of a Heck reaction (See, Jeffery,Tetrahedron (1996), 52, 10113 and Dieck and Heck, J. Org. Chem. (1975),40, 1083 and a NaOH promoted ester hydrolysis. The chloroortho-phosphate linker 21 was prepared via an oxidative coupling betweenthe bis-TMSE-phosphite (McCombie et al., J. Chem. Soc. (1945), 381) and2-hydroxybenzyl alcohol. Initially, problems were encountered in thelarge-scale preparation of linker 21 as it decomposed duringpurification giving low yields. The conditions were standardized byusing Et₃N as buffer to neutralize the acidity of silica gel to obtainthe linker in good quantities (88%). Heating a reaction mixtureconsisting of the acid 20, the linker 21, triethylamine anddiphenylphosphoryl azide under Curtius rearrangement conditions (Shippset al., J. Bioorg. Med. Chem. (1996), 4, 655) provided the desiredcarbamate 22 (58%), which was converted sequentially to the aldehyde 23(72%) and its corresponding thiosemicarbazone 24 in 63% yield. Theremoval of the 2-trimethylsilylethyl (TMSE) group in 24 was effectedcleanly with TFA (Chao et al., J. Org. Chem. (1994), 59, 6687) andprovided the 3-AP prodrug free acid 6, which was in turn converted tothe disodium salt 25 upon treatment with saturated sodium bicarbonatesolution and reverse phase column purification.

The other substituted ortho prodrugs were synthesized essentiallyfollowing the same route using appropriate phosphate-bearing substitutedbenzyl linkers such as 21. Coupling of these linkers to 25, followed byfunctional group manipulations furnished the corresponding prodrugs(FIG. 3). The synthesis evidences that the prodrugs of the presentinvention may be readily converted to their corresponding phosphatesalts. The water solubility of these phosphate salt compounds isexcellent and is significantly greater than corresponding non-prodrugforms. The solubility of parental 3-AP in aqueous solution is less than0.1 mg/ml, where as that of the prodrugs is between 16 and 35 mg/ml.

Having generally described the invention, reference is now made to thefollowing specific examples which are intended to illustrate preferredand other embodiments and comparisons. The included examples are not tobe construed as limiting the scope of this invention as is more broadlyset forth above and in the appended claims.

All reagents were purchased at commercial quality and used withoutfurther purification, and solvents were dried and/or distilled beforeuse where necessary. All NMR spectra (¹H, ¹³C, and ³¹P) were determinedon a Brucker AC300 spectrometer. Chemical shifts are measured in partsper million (ppm) relative to tetramethylsilane. Coupling constants arereported in hertz (Hz). Flash column chromatography (FCC) was performedwith Merck silica gel 60 (230-400 mesh), and pre-treated withtriethylamine for all trimethylsilylethyl (TMSE) protected compounds.Reversed phase column chromatography (RPCC) was packed with CAT gel(Waters, preparative C18 125 Å, 55-105 μm), eluting with milli-Qde-ionized water.

EXAMPLES 1-3 General Procedures for Preparation of the Nicotinic Acid(20) Example 1 Preparation of 2-chloronicotinic acid methyl ester (18)

To a mixture of 2-chloronicotinic acid (Aldrich, 100.0 g, 0.63 mol) in1,4-dioxane (500 mL) was added thionyl chloride (70 mL, 0.96 mol). Thesuspension was heated under reflux for 22 h with a gas trap to absorbhydrogen chloride gas. After evaporation of the solvent, the residue wasdissolved in methanol (300 mL). To the solution was added dropwisetriethylamine (TEA, 120 mL, 1.26 mol) at 0° C. over 2 h. The solventswere evaporated and the residue was suspended in ethyl acetate. Theprecipitate was removed by filtration. The filtrate was concentrated toafford the ester 18 (92.3 g, 86%) as an oil:

Rf(1:5 v/v ethyl acetate-hexane) 0.38.

¹H NMR (300 MHz, CDCl₃) δ 8.53 (dd, 4.8 Hz, 1H), 8.19 (dd, 7.6 Hz, 1H),7.37 (dd, 7.7 Hz, 1H) and 3.97 (s, 3H).

¹³C NMR (75 MHz, CDCl₃) δ 164.5, 151.6, 149.6, 140.0, 126.4, 121.9 and52.5.

Example 2 Preparation of 2-styrylnicotinic acid methyl ester (19)

To a solution of the ester 18 (48.8 g, 0.28 mol) in DMF (450 mL) wasadded styrene (165 mL, 1.42 mol), palladium acetate (6.5 g, 30 mmol),sodium acetate (47 g, 0.57 mol) and triphenyl phosphine (30 g, 0.11mol). The mixture was heated under reflux for 22 h. Thepalladium-catalyst was removed by filtration through a Celite pad. Thefiltrate was concentrated under reduced pressure, and the residue wasdissolved in a minimum amount of ethyl acetate. To the above solutionwas added hexane. After removal of the precipitate by filtration, thefiltrate was concentrated. The resulting crude material was purified byFCC (1:1 v/v ethyl acetate-hexane) to afford the ester 19 (55.0 g, 81%)as a light yellow oil:

Rf(1:5 v/v ethyl acetate-hexane) 0.41.

¹H NMR (300 MHz, CDCl₃) δ 8.70 (dd, 1H), 8.10 (dd, 1H), 8.16 (d, 1H),7.94 (d, 1H), 7.64 (d, 2H), 7.4-7.3 (m, 3H), 7.18 (dd, 1H) and 3.94 (s,3H).

¹³C NMR (75 MHz, CDCl₃) δ 166.7, 155.3, 152.0, 138.6, 136.7, 135.9,128.6, 128.5, 127.5, 124.8, 123.8, 121.3 and 52.4.

Example 3 Preparation of 2-styrylnicotinic acid (20)

A solution of the ester 19 (55.0 g, 0.23 mol) in THF (100 mL) wastreated with a 3 N NaOH solution (110 mL, 0.25 mol) for 21 h at ambienttemperature. After removal of solvents, the residue was taken up inwater and ethyl ether. The phases were separated, and the aqueous phasewas washed with ether (2×). The resulting aqueous phase was neutralizedwith a 2 N HCl solution, and the precipitate was then collected byfiltration to afford the acid 20 (50.2 g, 97%) as a cream solid:

¹H NMR (300 MHz, DMSO-d₆) δ 8.72 (dd, 1H), 8.19 (dd, 1H), 8.10 (d, 1H),7.86 (d, 1H), 7.62 (d, 2H) and 7.4−7.3 (m, 4H).

¹³C NMR (75 MHz, DMSO-d₆) δ 167.9, 153.7, 151.8, 138.6, 136.4, 134.5,128.9, 128.7, 127.2, 125.3 and 122.1.

EXAMPLES 4-5 General Procedures for Preparation of the Phosphate Linkers(21, 26 a-k) Example 4 Preparation ofbis(2-trimethylsilylethyl)phosphite (TMSE-phosphite)

To a solution of 2-(trimethylsilyl)ethanol (Aldrich, 25.0 g, 0.21 mol)in ethyl ether (200 mL) containing pyridine (11.4 mL, 0.14 mol) wasadded phosphorus trichloride (6.2 mL, 70 mmol) in one portion at −78° C.The reaction mixture was kept for 5 min while stirring, and then dilutedwith ethyl ether (500 mL). After warming to ambient temperature, themixture was stirred for 18 h continually. The precipitate was removed byfiltration, and the filtrate was then bubbled by ammonia gas for 10 min.The precipitate was removed by filtration through a Celite pad, and thefiltrate was concentrated to afford TMSE-phosphite (20.7 g, 99%) as acolorless oil:

¹H NMR (300 MHz, CDCl₃) δ 6.76 (d, 1H), 4.13 (m, 4H), 1.07 (m, 4H) and0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 64.0 (d), 19.6 (d) and −1.6 (d).

³¹P NMR (121 MHz, CDCl₃) δ 18.5.

Example 5 Preparation of 2-(TMSE-phosphonooxy)benzyl alcohols (21, 26a-k)

General Procedure. To a solution of the corresponding 2-hydroxybenzylalcohol (10 mmol) in acetonitrile (40 mL) was addedN,N′-diisopropylethylamine (DIEA, 11 mmol), 4-dimethylaminopyridine(DMAP, 1 mmol), and carbon tetrachloride (50 mmol). While stirring at−30° C., to the solution was added bis(2-trimethylsilylethyl)phosphite(stored in refrigerator, 11 mmol) immediately. After warming to ambienttemperature, the reaction mixture was stirred for 3 h. The solvents wereevaporated under reduced pressure, and the residual product was purifiedby FCC (1:1 v/v ethyl acetate-hexane) to afford the correspondingTMSE-protected phosphate linker (21, 26 a-k).

2-Bis(2-trimethylsilylethyl)phosphonooxy-5-chlorobenzyl alcohol (21).

Following the above procedure, 5-chloro-2-hydroxybenzyl alcohol (5.0 g,32 mmol) gave 21 (12.2 g, 88%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 7.33 (d, 1H), 7.11 (m, 1H), 7.02 (m, 1H), 4.49(s, 2H), 4.12 (m, 4H), 1.00 (m, 4H) and −0.07 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 146.5 (d), 134.7 (d), 130.8, 130.2, 128.6,122.0 (d), 67.7 (d), 59.4, 19.5 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 6.1.

2-Bis(2-trimethylsilylethyl)phosphonooxy-5-fluorobenzyl alcohol (26 a).

Following the above procedure, 5-fluoro-2-hydroxybenzyl alcohol (17.0 g,119 mmol) gave 26 a (31.7 g, 62%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 7.2−7.1 (m, 1H), 7.0−6.9 (m, 1H), 4.63 (s,1H), 4.3−4.1 (m, 4H), 1.2−1.1 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 159.8 (d), 143.7 (dd), 135.2 (dd), 121.8 (dd),116.4 (d), 115.0 (d), 67.6 (d), 59.4, 19.5 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 6.4.

¹⁹F NMR (282 MHz, CDCl₃) δ−59.8.

2-Bis(2-trimethylsilylethyl)phosphonooxy-5-nitrobenzyl alcohol (26 b).

Following the above procedure, 2-hydroxy-5-nitrobenzyl alcohol (4.5 g,27 mmol) gave 26 b (6.4 g, 53%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 8.14 (m, 1H), 7.81 (m, 1H), 7.14 (m, 1H), 4.48(s, 2H), 4.06 (m, 4H), 0.90 (m, 4H) and −0.20 (m, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 152.0 (d), 144.6, 134.7 (d), 123.7, 123.4,119.8, 67.9 (d), 58.4, 19.3 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 4.4.

2-Bis(2-trimethylsilylethyl)phosphonooxy-5-methoxybenzyl alcohol (26 c).

Following the above procedure, 2-hydroxy-5-methoxybenzyl alcohol (11.0g, 25 mmol) gave 26 c (7.7 g, 70%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 7.06 (dd, 1H), 6.94 (d, 1H), 6.74 (dd, 1H),4.57 (s, 2H), 4.3−4.1 (m, 4H), 3.74 (s, 3H), 1.1−1.0 (m, 4H) and 0.0 (m,18H).

¹³C NMR (75 MHz, CDCl₃) δ 157.1, 141.7 (d), 134.0 (d), 121.9 (d), 125.3,114.5, 67.5 (d), 60.2, 55.6, 19.6 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 6.9.

2-Bis(2-trimethylsilylethyl)phosphonooxy-5-trifluoromethoxybenzylalcohol (26 d).

Following the above procedure, 2-hydroxy-5-trifluoromethoxybenzylalcohol (1.9 g, 9.1 mmol) gave 26 d (3.3 g, 62%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 7.20 (d, 1H), 7.19 (dd, 1H), 7.09 (dd, 1H),4.61 (s, 2H), 4.24 (m, 4H), 1.08 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 146.4 (dd), 135.0 (d), 123.0, 122.1, 121.4,67.8 (d), 59.6, 19.6 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 6.2.

¹⁹F NMR (282 MHz, CDCl₃) δ−58.7.

2-Bis(2-trimethylsilylethyl)phosphonooxy-5-trifluoromethylbenzyl alcohol(26 e).

Following the above procedure, 2-hydroxy-5-trifluoromethylbenzyl alcohol(4.1 g, 22 mmol) gave 26 e (7.9 g, 77%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 7.72 (br s, 1H), 7.51 (dd, 1H), 7.29 (d, 1H),4.66 (s, 2H), 4.23 (m, 4H), 1.09 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 150.6 (d), 133.8 (d), 127.7 (d), 126.1, 121.3(d), 68.0 (d), 59.6, 19.6 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.8.

¹⁹F NMR (282 MHz, CDCl₃) δ−62.9.

2-Bis(2-trimethylsilylethyl)phosphonooxy-3,5-dichlorobenzyl alcohol (26f).

Following the above procedure, 3,5-dichloro-2-hydroxybenzyl alcohol (4.6g, 24 mmol) gave 26 f (7.2 g, 63%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 7.34 (d, 1H), 7.32 (dd, 1H), 4.56 (s, 2H),4.25 (m, 4H), 1.08 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 149.9, 143.3 (d), 136.9 (d), 131.2 (d), 129.9,129.5, 127.6 (d), 68.3 (d), 59.8, 19.5 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.7.

2-Bis(2-trimethylsilylethyl)phosphonooxy-4,5-dichlorobenzyl alcohol (26g).

Following the above procedure, 4,5-dichloro-2-hydroxybenzyl alcohol (3.6g, 18 mmol) gave 26 g (5.2 g, 59%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 7.53 (s, 1H), 7.28 (s, 1H), 4.55 (s, 2H), 4.21(m, 4H), 1.08 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 146.6 (d), 133.5 (d), 131.9 (d), 131.6, 129.5(d), 123.0 (d), 68.1 (d), 59.1, 19.6 (d) and −1.5.

³¹P NMR (121 MHz, CDCl₃) δ 6.0.

2-Bis(2-trimethylsilylethyl)phosphonooxy-5,6-dichlorobenzyl alcohol (26h).

Following the above procedure, 5,6-dichloro-2-hydroxybenzyl alcohol (4.8g, 25 mmol) gave 26 h (8.6 g, 73%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 7.35 (d, 1H), 7.04 (dd, 1H), 4.76 (s, 2H),4.22 (m, 4H), 1.08 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 147.6 (d), 134.7, 133.5 (d), 130.6 (d), 129.9,120.8 (d), 68.1 (d), 57.3, 19.6 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 6.4;

2-Bis(2-trimethylsilylethyl)phosphonooxy-3-methylbenzyl alcohol (26 i).

Following the above procedure, 2-hydroxy-3-methylbenzyl alcohol (2.0 g,14 mmol) gave 26 i (1.7 g, 88%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 7.16 (m, 1H), 7.0−6.9 (m, 2H), 4.48 (s, 2H),4.13 (m, 4H), 2.22 (s, 3H), 0.97 (m, 4H) and −0.09 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 146.9 (d), 133.5 (d), 131.0, 130.4 (d), 129.4,125.6 (d), 67.6 (d), 60.1, 19.5 (d), 16.8 and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 6.9.

2-Bis(2-trimethylsilylethyl)phosphonooxy-4-chlorobenzyl alcohol (26 j).

Following the above procedure, 4-chloro-2-hydroxybenzyl alcohol (4.2 g,26 mmol) gave 26 j (9.6 g, 84%) as an oil:

R_(f) (4:1 v/v ethyl acetate-hexane) 0.67.

¹H NMR (300 MHz, CDCl₃) δ 7.27 (d, 1H), 7.2-7.1 (m, 2H), 4.55 (s, 2H),4.21 (m, 4H), 1.07 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 148.5 (d), 133.9, 131.7 (d), 131.5, 126.0,121.4 (d), 67.9 (d), 59.4, 19.5 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.8.

2-Bis(2-trimethylsilylethyl)phosphonooxy-4-methoxybenzyl alcohol (26 k).

Following the above procedure, 2-hydroxy-4-methoxybenzyl alcohol (2.7 g,17 mmol) gave 26 k (2.5 g, 33%) as an oil:

Rf (4:1 v/v ethyl acetate-hexane) 0.70.

¹H NMR (300 MHz, CDCl₃) δ 7.29 (d, 1H), 6.8−6.7 (m, 2H), 4.53 (s, 2H),4.22 (m, 4H), 3.75 (s, 3H), 1.09 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 160.1 (d), 149.1 (d), 131.9, 125.3 (d), 111.2,107.3 (d), 67.6 (d), 59.7, 55.5, 19.6 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 6.4.

EXAMPLES 6-10 General Procedures for Preparation of the 3-AP Prodrugs(25, 30 a-k) Example 6 Preparation of (2-styrylpyridin-3-yl)carbamicacid 2-(TMSE-phosphonooxy)benzyl esters (22, 27 a-k) (CurtiusRearrangement)

General Procedure. To a solution of 2-styrylnicotinic acid (20, 20 mmol)in benzene (100 mL) containing triethylamine (TEA, 32 mmol) was addeddiphenylphosphorylazide (32 mmol). The solution was heated at reflux for10 min, and the corresponding TMSE-protected phosphate linker (21 or 26a-k, 20 mmol) was then added. The reaction mixture was kept under refluxfor 3 h. Next, the solvents were evaporated under reduced pressure. Theresidual product was purified by FCC (1:4 v/v ethyl acetate-hexane) toafford the corresponding carbamate (22 or 27 a-k).

(2-Styrylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5-chlorobenzyl ester (22).

Following the above procedure, 21 (9.4 g, 21 mmol) gave 22 (10.6 g, 58%)as an oil:

¹H NMR (300 MHz, CDCl₃) δ 8.23 (d, 1H), 7.92 (br s, 1H), 7.56 (d, 1H),7.4−7.0 (m, 11H), 5.11 (s, 2H), 4.10 (m, 4H), 0.94 (m, 4H) and −0.14 (s,18H).

¹³C NMR (75 MHz, CDCl₃) δ 153.7, 151.9, 147.3 (d), 146.8, 145.2, 136.5,134.8, 131.4, 130.2, 129.5, 129.3, 129.2, 128.5, 128.3, 127.2, 122.3,121.3, 121.2, 67.4 (d), 61.6, 19.4 (d) and −1.7.

³¹P NMR (121 MHz, CDCl₃) δ 5.1.

Mass Calcd. For C₃₁H₄₂ClN₂O₆PSi₂: 661.277; Found: 661.2

(2-Styrylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5-fluorobenzyl ester (27 a).

Following the above procedure, the cruse carbamate 27 a obtained from 26a (31.0 g, 73 mmol) was directly used for the further reaction withoutpurification.

(2-Styrylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5-nitrobenzyl ester (27 b).

Following the above procedure, 26 b (4.3 g, 9.6 mmol) gave 27 b (2.3 g,35%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 8.3−7.0 (m, 14H), 5.21 (s, 2H), 4.16 (m, 4H),0.99 (m, 4H) and −0.12 (m, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 153.5, 153.2 (d), 149.9, 145.6, 144.3, 136.4,135.1, 131.1, 129.3, 129.0 (d), 128.5, 128.4, 127.2, 124.9, 124.8,122.4, 120.9, 120.3, 68.0 (d), 62.1, 19.5 (d), 17.1 and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 4.5.

(2-Styrylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5-methoxybenzyl ester (27 c).

Following the above procedure, the cruse carbamate 27 c obtained from 26c (6.0 g, 26 mmol) was directly used for the further reaction withoutpurification.

(2-Styrylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5-trifluoromethoxybenzyl ester(27 d).

Following the above procedure, 26 d (1.9 g, 8.5 mmol) gave 27 d (3.4 g,83%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 8.39 (dd, 1H), 8.13 (br s, 1H), 7.74 (d, 1H),7.60 (m, 2H), 7.4 1 (dd, 1H), 7.4−7.1 (m, 7H), 5.30 (s, 2H), 4.27 (m,4H), 1.10 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 153.4, 147.3 (d), 145.7, 145.4, 136.6, 135.4,131.2, 129.8, 129.7, 129.2 (d), 128.6, 128.5, 127.4, 127.0, 125.2,122.7, 122.5, 122.2, 121.5, 120.8, 120.0 (d), 118.6, 67.6 (d), 62.0,19.6 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.3.

¹⁹F NMR (282 MHz, CDCl₃) δ−58.7.

(2-Styrylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5-trifluoromethylbenzyl ester(27 e).

Following the above procedure, 26 e (5.1 g, 11 mmol) gave 27 e (5.3 g,71%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 8.40 (dd, 1H), 8.14 (br s, 1H), 7.74 (d, 2H),7.6−7.5 (m, 4H), 7.4−7.1 (m, 8H), 5.29 (s, 2H), 4.29 (m, 4H), 1.11 (m,4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 153.4, 145.4 (m), 136.5, 135.4, 131.1, 129.7,128.6, 128.5, 128.2, 128.1, 127.4, 127.1 (d), 122.5, 120.8, 120.5, 120.0(d), 67.7 (d), 61.9, 19.6 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.0.

¹⁹F NMR (282 MHz, CDCl₃) δ−62.7.

(2-Styrylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-3,5-dichlorobenzyl ester (27f).

Following the above procedure, 26 f (6.3 g, 13 mmol) gave 27 f (7.1 g,76%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 8.39 (dd, 1H), 8.11 (br s, 1H), 7.74 (d, 1H),7.59 (br d, 2H), 7.4−7.2 (m, 8H), 7.18 (dd, 2H), 5.35 (s, 2H), 4.29 (m,4H), 1.11 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 153.3, 145.4 (d), 136.5, 135.4, 131.9 (d),131.1, 131.0, 130.2, 129.8, 129.7, 128.7, 128.6, 128.3, 128.0 (d),127.4, 122.5, 120.8, 67.9 (d), 62.2, 19.5 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.0.

(2-Styrylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-4,5-dichlorobenzyl ester (27g).

Following the above procedure, 26 g (11.3 g, 50 mmol) gave 27 g (17.4 g,75%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 8.38 (dd, 1H), 8.12 (br s, 1H), 7.74 (d, 1H),7.60 (dd, 2H), 7.53 (s, 1H), 7.51 (d, 1H), 7.4−7.2 (m, 6H), 7.17 (dd,2H), 5.23 (s, 2H), 4.27 (m, 4H), 1.10 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 153.4, 147.7 (d), 145.4, 136.6, 135.4, 133.1,131.3, 131.2, 128.9, 128.6, 128.5, 127.7 (d), 127.4, 122.5, 120.8, 67.8(d), 61.5, 19.6 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.2.

(2-Styrylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5,6-dichlorobenzyl ester (27h).

Following the above procedure, 26 h (6.2 g, 28 mmol) gave 27 h (9.6 g,75%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 8.38 (dd, 1H), 8.20 (br s, 1H), 7.73 (d, 1H),7.60 (br d, 2H), 7.48 (d, 1H), 7.4−7.2 (m, 7H), 7.18 (dd, 2H), 5.48 (s,2H), 4.28 (m, 4H), 1.10 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 153.4, 149.2 (d), 145.2, 136.5, 135.4, 134.7,131.3, 131.0, 129.8, 129.4, 128.6, 128.5, 127.4, 127.3 (d), 122.5,120.7, 119.6 (d), 67.7 (d), 59.9, 19.6 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.0.

(2-Styrylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-3-methylbenzyl ester (27 i).

Following the above procedure, 26 i (1.4 g, 6.2 mmol) gave 27 i (1.5 g,53%) as an orange oil:

¹H NMR (300 MHz, CDCl₃) δ 8.23 (dd, 1H), 7.97 (br s, 1H), 7.66 (m, 1H),7.58 (d, 1H), 7.4−6.9 (m, 10H), 5.27 (s, 2H), 4.11 (m, 4H), 2.27 (s,3H), 0.94 (m, 4H) and −0.11 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 153.9, 147.6 (d), 146.4, 145.1, 136.6, 134.7,131.7, 131.6, 131.0, 130.8 (d), 128.5, 128.4 (d), 128.3, 127.6, 127.3,125.3, 122.3, 121.2, 67.1 (d), 62.9, 19.5 (d), 17.1 and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.6.

Mass Calcd. For C₃₂H₄₅ N₂O₆PSi₂: 640.859; Found: 640.2

(2-Styrylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-4-chlorobenzyl ester (27 j).

Following the above procedure, 26 j (9.3 g, 21 mmol) gave 27j (12.6 g,87%) as an oil:

Rf (1:1 v/v ethyl acetate-hexane) 0.82.

¹H NMR (300 MHz, CDCl₃) δ 8.28 (dd, 1H), 8.15 (br s, 1H), 7.72 (d, 1H),7.60 (d, 2H), 7.4−7.3 (m, 7H), 7.2−7.1 (m, 2H), 5.26 (s, 2H), 4.28 (m,4H), 1.11 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 153.6, 149.7 (d), 145.2, 136.6, 135.3, 135.1,131.4, 131.3, 128.6, 128.5, 127.4, 125.9 (d), 125.4, 122.4, 120.9 (d),120.8, 67.6 (d), 62.1, 19.5 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 65.1.

(2-Styrylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-4-methoxybenzyl ester (27 k).

Following the above procedure, 26 k (2.8 g, 6.5 mmol) gave 27 k (3.7 g,86%) as an oil:

Rf(1:1 v/v ethyl acetate-hexane) 0.50.

¹H NMR (300 MHz, CDCl₃) δ 8.36 (dd, 1H), 8.18 (br s, 1H), 7.72 (d, 1H),7.4−7.3 (m, 6H), 7.62 (d, 2H), 7.2−7.1 (m, 1H), 6.97 (m, 1H), 6.71 (dd,1H), 5.24 (s, 2H), 4.29 (m, 4H), 3.80 (s, 3H), 1.11 (m, 4H) and 0.0 (s,18H).

¹³C NMR (75 MHz, CDCl₃) δ 160.5, 153.9, 150.0 (d), 145.0, 136.7, 135.1,131.9, 131.6, 130.0, 128.6, 128.4, 127.4, 122.4, 120.9, 119.2 (d),110.4,67.3 (d), 62.4, 55.5, 19.6 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.2.

Example 7 Preparation of (2-formylpyridin-3-yl)carbamic acid2-(TMSE-phosphonooxy)benzyl esters (23, 28 a-k) (ozonolysis)

General Procedure. The corresponding 2-styrylpyridine (22 or 27 a-k, 10mmol) was dissolved in dichloromethane (50 mL) and ethanol (40 mL). Thelight yellow solution was ozonized at −50° C. till the solution turnedto light blue. Nitrogen gas was bubbled through the solution for 30 minto expel excess ozone. To the solution was then added dimethyl sulfide(5 mL), and the mixture was stirred for 2 h at room temperature. Thesolvent was evaporated under reduced pressure, and the residual productwas purified by FCC (1:9 v/v ethyl acetate-hexane) to afford thecorresponding pyridine-2-carboxaldehyde (23 or 28 a-k).

(2-Formylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5-chlorobenzyl ester (23).

Following the above procedure, 22 (2.4 g, 3.7 mmol) gave 23 (1.6 g, 72%)as an oil:

¹H NMR (300 MHz, CDCl₃) δ 10.38 (br s, 1H), 9.90 (s, 1H), 8.67 (d, 1H),8.28 (dd, 1H), 7.4−7.3 (m, 2H), 7.23 (dd, 1H), 7.13 (dd, 1H), 5.14 (s,2H), 4.12 (m, 4H), 0.97 (m, 4H) and −0.14 (m, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 197.0, 152.9, 147.3 (d), 143.7, 138.3, 136.7,130.1, 129.6, 129.5, 128.6, 128.5, 126.2, 121.3, 67.4 (d), 61.6, 19.4(d) and −1.7.

³¹P NMR (121 MHz, CDCl₃) δ 5.1.

(2-Formylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5-fluorobenzyl ester (28 a).

Following the above procedure, the crude 27 a (31.0 g, 73 mmol) gave 28a (26.9 g, 64%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 10.58 (s, 1H), 10.10 (s, 1H), 8.86 (d, 1H),8.48 (dd, 1H), 7.52 (m, 1H), 7.4−7.3 (m, 1H), 7.21 (dd, 1H), 7.1-6.9 (m,1H), 5.30 (s, 2H), 4.4−4.2 (m, 4H), 1.2−1.0 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 197.1, 159.3 (d), 152.9, 144.5 (dd), 143.7,143.5, 138.4, 136.8, 121.4 (dd), 116.2 (d), 115.9 (d), 67.3 (d), 61.8,19.5 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.5.

¹⁹F NMR (282 MHz, CDCl₃) δ−59.3.

(2-Formylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5-nitrobenzyl ester (28 b).

Following the above procedure, 27 b (4.2 g, 9.4 mmol) gave 28 b (2.8 g,50%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 10.42 (br s, 1H), 9.89 (s, 1H), 8.64 (d, 1H),8.28 (dd, 1H), 8.21 (d, 1H), 8.05 (dd, 1H), 7.47 (d, 1H), 7.33 (dd, 1H),5.21 (s, 2H), 4.17 (m, 4H), 0.98 (m, 4H) and −0.13 (m, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 197.0, 153.5 (d), 152.7, 144.3, 143.9, 138.1,136.8, 128.6, 128.3 (d), 126.2, 125.4, 125.2, 120.3, 67.9 (d), 61.4,19.5 (d) and −1.7.

³¹P NMR (121 MHz, CDCl₃) δ 4.5.

(2-Formylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5-methoxybenzyl ester (28 c).

Following the above procedure, the crude 27 c (7.5 g, 17 mmol) gave 28 c(9.8 g, 73%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 10.37 (s, 1H), 9.93 (s, 1H), 8.71 (d, 1H),8.30 (d, 1H), 7.34 (dd, 1H), 7.19 (d, 1H), 6.85 (d, 1H), 6.70 (d, 1H),5.18 (s, 2H), 4.2−4.0 (m, 4H), 3.66 (s, 3H), 1.1−0.9 (m, 4H) and 0.0 (s,18H).

¹³C NMR (75 MHz, CDCl₃) δ 196.9, 156.4, 153.1, 143.6, 142.4 (d), 138.5,136.7, 128.6, 127.7 (d), 126.2, 120.9, 115.1, 114.3, 67.1 (d), 62.3,55.5, 19.4 (d) and −1.7.

³¹P NMR (121 MHz, CDCl₃) δ 5.8.

(2-Formylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5-trifluoromethoxybenzyl ester(28 d).

Following the above procedure, 27 d (4.7 g, 6.6 mmol) gave 28 d (3.2 g,75%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 10.54 (br s, 1H), 10.06 (s, 1H), 8.82 (d, 1H),8.44 (dd, 1H), 7.48 (dd, 1H), 7.44 (dd, 1H), 7.32 (d, 1H), 7.25 (s, 1H),7.2−7.1 (m, 1H), 5.30 (s, 2H), 4.26 (m, 4H), 1.10 (m, 4H) and 0.0 (s,18H).

¹³C NMR (75 MHz, CDCl₃) δ 197.2, 153.0, 147.1 (d), 145.7, 143.9, 138.4,136.9, 129.8, 128.8, 128.7, 126.4, 122.6, 122.2, 121.3, 120.0, 119.9,67.6 (d), 61.8, 19.5 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.3.

¹⁹F NMR (282 MHz, CDCl₃) δ−58.8.

(2-Formylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5-trifluoromethylbenzyl ester(28 e).

Following the above procedure, 27 e (12.2 g, 18 mmol) gave 28 e (6.9 g,63%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 10.54 (br s, 1H), 10.06 (d, 1H), 8.82 (br d,1H), 8.44 (dd, 1H), 7.72 (br s, 1H), 7.6−7.5 (m, 2H), 7.48 (dd, 1H),7.3−7.1 (m, 2H), 5.33 (s, 2H), 4.27 (m, 4H), 1.10 (m, 4H) and 0.0 (s,18H).

¹³C NMR (75 MHz, CDCl₃) δ 197.1, 153.0, 151.5 (d), 143.9, 138.4, 136.9,129.8, 129.7, 128.7, 127.7, 127.6, 127.3 (d), 127.1, 127.0, 126.4,126.3, 125.2, 120.3 (d), 120.0 (d), 67.7 (d), 61.8, 19.6 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 4.9.

¹⁹F NMR (282 MHz, CDCl₃) δ−62.7.

(2-Formylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-3,5-dichlorobenzyl ester (28f).

Following the above procedure, 27 f (8.0 g, 12 mmol) gave 28 f (5.1 g,71%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 10.54 (br s, 1H), 10.05 (s, 1H), 8.79 (d, 1H),8.42 (dd, 1H), 7.46 (dd, 1H), 7.35 (dd, 2H), 7.24 (s, 1H), 5.38 (s, 2H),4.27 (m, 4H), 1.12 (m, 4H) and 0.0 (m, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 197.2, 152.8, 149.9, 143.9, 143.6 (d), 138.4,136.8, 131.8 (d), 131.0 (d), 130.1, 128.7, 128.0 (d), 127.8 (d), 126.3,120.0 (d), 67.8 (d), 62.0, 19.6 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.1.

(2-Formylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-4,5-dichlorobenzyl ester (28g).

Following the above procedure, 27 g (17.4 g, 25 mmol) gave 28 g (13.4 g,86%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 10.51 (br s, 1H), 10.05 (s, 1H), 8.79 (d, 1H),8.42 (dd, 1H), 7.53 (dd, 1H), 7.5−7.4 (m, 1H), 7.24 (s, 1H), 6.96 (dd,1H), 5.23 (s, 2H), 4.28 (m, 4H), 1.10 (m, 4H) and 0.0 (m, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 197.2, 152.9, 147.4 (d), 144.0, 138.3, 136.9,133.1, 131.0, 128.8 (d), 128.7, 127.2 (d), 126.3, 122.2, 122.0, 67.8(d), 61.3, 19.6 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.1.

(2-Formylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-5,6-dichlorobenzyl ester (28h).

Following the above procedure, 27 h (9.5 g, 14 mmol) gave 28 h (6.5 g,77%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 10.44 (br s, 1H), 10.05 (s, 1H), 8.86 (d, 1H),8.44 (dd, 1H), 7.50 (d, 1H), 7.47 (d, 1H), 7.41 (d, 1H), 7.38 (m, 1H),5.46 (s, 2H), 4.25 (m, 4H), 1.10 (m, 4H) and 0.0 (m, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 197.0, 153.0, 149.9, 149.2 (d), 143.8, 138.5,136.8, 135.0, 131.2, 129.8, 129.6, 128.7, 126.6 (d), 126.3, 119.5 (d),67.7 (d), 59.9, 19.5 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 4.9.

(2-Formylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-3-methylbenzyl ester (28 i).

Following the above procedure, 27 i (1.5 g, 2.2 mmol) gave 28 i (1.1 g,86%) as an oil:

¹H NMR (300 MHz, CDCl₃) δ 10.35 (br s, 1H), 9.92 (s, 1H), 8.71 (d, 1H),8.28 (dd, 1H), 7.32 (dd, 1H), 7.16 (d, 1H), 7.05 (d, 1H), 6.96 (dd, 1H),5.31 (s, 2H), 4.12 (m, 4H), 2.28 (s, 3H), 0.97 (m, 4H) and −0.12 (m,18H).

¹³C NMR (75 MHz, CDCl₃) δ 196.9, 153.2, 147.2 (d), 143.6, 138.6, 136.6,131.6, 130.9 (d), 128.6, 128.0 (d), 127.4, 126.2, 125.3, 67.1 (d), 62.9,19.4 (d), 17.0 and −1.7.

³¹P NMR (121 MHz, CDCl₃) δ 5.8.

(2-Formylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-4-chlorobenzyl ester (28 j).

Following the above procedure, 27 j (12.2 g, 19 mmol) gave 28 j (8.9 g,80%) as an oil:

Rf (1:1 v/v ethyl acetate-hexane) 0.66.

¹H NMR (300 MHz, CDCl₃) δ 10.49 (br s, 1H), 10.04 (s, 1H), 8.80 (d, 1H),8.42 (dd, 1H), 7.5−7.4 (m, 3H), 7.16 (dd, 1H), 5.26 (s, 2H), 4.27 (m,4H), 1.11 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, CDCl₃) δ 197.1, 153.1, 149.3 (d), 143.8, 138.5, 136.8,135.0, 131.0, 128.7, 126.3, 125.3 (d), 125.2, 120.5 (d), 67.5 (d), 61.9,19.5 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.0.

(2-Formylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl)phosphonooxy-4-methoxybenzyl ester (28 k).

Following the above procedure, 27 k (5.1 g, 8.0 mmol) gave 28 k (2.7 g,58%) as an oil:

Rf (1:1 v/v ethyl acetate-hexane) 0.44.

¹H NMR (300 MHz, CDCl₃) δ 10.50 (br s, 1H), 10.05 (s, 1H), 8.84 (d, 1H),8.42 (dd, 1H), 7.46 (dd, 1H), 7.36 (dd, 1H), 7.01 (s, 1H), 6.71 (dd,1H), 5.24 (s, 2H), 4.27 (m, 4H), 3.80 (s, 3H), 1.10 (m, 4H) and 0.0 (s,18H).

¹³C NMR (75 MHz, CDCl₃) δ 197.0, 160.9, 153.4, 150.2 (d), 143.6, 138.7,136.7, 131.7, 128.7, 126.3, 118.5 (d), 110.6, 106.1 (d), 67.3 (d), 62.4,55.5, 19.5 (d) and −1.6.

³¹P NMR (121 MHz, CDCl₃) δ 5.0.

Example 8 Preparation of pyridine-2-carboxaldehyde thiosemicarbazones(24, 29 a-k)

General Procedure. The corresponding pyridine-2-formaldehyde (23 or 28a-k, 10 mmol) was dissolved in ethanol-water (2:1 v/v, 150 mL). To thesolution was added thiosemicarbazide (11 mmol). The solution was stirredfor 30 min at ambient temperature. After addition of water (50 mL), thereaction mixture was stirred vigorously for 2 h at room temperature. Theyellow precipitate was collected by filtration, washed withethanol-water (1:4 v/v) and dried in vacuum to afford the correspondingpyridine-2-carboxaldehyde thiosemicarbazone (24, 29 a-k).

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid 2-bis(2-trimethylsilyl ethyl) phosphonooxy-5-chlorobenzyl ester (24).

Following the above procedure, 23 (6.9 g, 12 mmol) gave 24 (4.9 g, 63%)as a yellow solid:

¹H NMR (300 MHz, DMSO-d₆) δ 11.77 (br s, 1H), 10.03 (br s, 1H), 8.40(dd, 1H), 8.28 (br s, 1H), 8.26 (s, 1H), 7.94 (br s, 1H), 7.5−7.4 (m,4H), 5.20 (s, 2H), 4.06 (m, 4H), 0.98 (m, 4H) and −0.03 (m, 18H).

¹³C NMR (75 MHz, DMSO-d₆) δ 178.5, 153.4, 148.0 (d), 144.2, 142.9,141.0, 133.9, 129.5 (d), 128.9, 128.4, 128.0, 124.4, 121.6, 65.1 (d),61.2, 18.9 (d) and −1.5.

³¹P NMR (121 MHz, DMSO-d₆) δ 9.8.

Mass Calcd. For C₂₅H₃₉ClN₅O₆PSSi₂: 660.267; Found: 660.2

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl) phosphonooxy-5-fluorobenzyl ester (29 a).

Following the above procedure, 28 a (14.3 g, 25 mmol) gave 29 a (12.9 g,80%) as a yellow solid:

¹H NMR (300 MHz, DMSO-d₆) δ 11.85 (s, 1H), 10.13 (s, 1H), 8.45 (d, 1H),8.26 (s, 1H), 7.5−7.1 (m, 5H), 5.21 (s, 2H), 4.2−4.0 (m, 4H), 1.0−0.9(m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, DMSO-d₆) δ 178.6, 159.8, 156.0, 153.4, 145.1 (d),143.5, 141.5, 140.4, 134.2, 129.5, 124.5, 121.4 (d), 115.5, 64.4 (d),61.3, 18.9 (d) and −1.6.

³¹P NMR (121 MHz, DMSO-d₆) δ 10.5.

¹⁹F NMR (282 MHz, DMSO-d₆) δ−62.5.

Mass Calcd. For C₂₅H₃₉FN₅O₆PSSi₂: 643.813 Found: 644.2

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl) phosphonooxy-5-nitrobenzyl ester (29 b).

Following the above procedure, 28 b (1.6 g, 2.7 mmol) gave 29 b (1.3 g,77%) as a yellow solid:

¹H NMR (300 MHz, DMSO-d₆) δ 11.86 (br s, 1H), 10.14 (br s, 1H), 8.4−8.2(m, 4H), 7.87 (br s, 2H), 7.64 (m, 1H), 7.52 (m, 1H), 5.26 (s, 2H), 4.05(m, 4H), 0.97 (m, 4H) and −0.03 (m, 18H).

¹³C NMR (75 MHz, DMSO-d₆) δ 178.6, 155.3 (d), 153.4, 143.7, 142.7,140.7, 134.1, 128.1 (d), 125.2, 124.6, 124.5, 120.1, 64.8 (d), 61.2,18.9 (d) and −1.5.

³¹P NMR (121 MHz, DMSO-d₆) δ 9.2.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl) phosphonooxy-5-methoxybenzyl ester (29 c).

Following the above procedure, 28 c (5.0 g, 8.8 mmol) gave 29 c (4.4 g,77%) as a yellow solid:

¹H NMR (300 MHz, DMSO-d₆) δ 11.85 (s, 1H), 10.08 (s, 1H), 8.41 (d, 1H),8.35 (d, 1H), 8.30 (s, 1H), 8.03 (s, 2H), 7.51 (dd, 1H), 7.26 (d, 1H),7.01 (d, 1H), 6.90 (dd, 1H), 5.26 (s, 2H), 4.2−4.0 (m, 4H), 3.75 (s,3H), 1.1−0.9 (m, 4H) and 0.0 (s, 18H).

¹³C NMR (75 MHz, DMSO-d₆) δ 178.7, 155.7, 153.6, 143.9, 142.6(d), 134.2,129.4, 128.4 (d), 124.5, 121.1, 114.1, 113.9, 64.9 (d), 61.9, 55.6, 19.1(d) and −1.4.

³¹P NMR (121 MHz, DMSO-d₆) δ 10.3.

Mass Calcd. For C₂₆H₄₂ N₅O₇PSSi₂: 655.848 Found: 656.2

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl) phosphonooxy-5-trifluoromethoxybenzyl ester(29 d).

Following the above procedure, 28 d (2.5 g, 3.9 mmol) gave 29 d (1.9 g,68%) as a yellow solid:

¹H NMR (300 MHz, DMSO-d₆) δ 11.81 (s, 1H), 10.04 (br s, 1H), 8.42 (d,1H), 8.26 (s, 1H), 7.95 (br s, 1H), 7.5−7.2 (m, 4H), 5.24 (s, 2H), 4.07(m, 4H), 0.96 (m, 4H) and −0.04 (m, 18H).

¹³C NMR (75 MHz, DMSO-d₆) δ 178.4, 153.4, 147.8 (d), 144.1, 144.0,133.9, 129.4 (d), 124.4, 121.9, 121.7, 121.6, 121.4, 118.3, 65.1 (d),61.2, 18.9 (d) and −1.6.

³¹P NMR (121 MHz, DMSO-d₆) δ 9.8.

¹⁹F NMR (282 MHz, DMSO-d₆) δ−53.0.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl) phosphonooxy-5-trifluoromethylbenzyl ester(29 e).

Following the above procedure, 28 e (5.3 g, 8.5 mmol) gave 29 e (3.6 g,61%) as a yellow solid:

¹H NMR (300 MHz, DMSO-d₆) δ 11.81 (s, 1H), 10.03 (br s, 1H), 8.42 (d,1H), 8.4−8.3 (m, 2H), 8.26 (s, 1H), 7.89 (br s, 1H), 7.79 (s, 1H), 7.75(d, 2H), 7.56 (d, 2H), 7.49 (dd, 1H), 5.27 (s, 2H), 4.06 (m, 4H), 0.97(m, 4H) and −0.04 (m, 18H).

³¹P NMR (121 MHz, DMSO-d₆) δ 9.5.

¹⁹F NMR (282 MHz, DMSO-d₆) δ−56.2.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl) phosphonooxy-3,5-dichlorobenzyl ester (29f).

Following the above procedure, 28 f (4.8 g, 7.7 mmol) gave 29 f (4.6 g,85%) as a yellow solid:

¹H NMR (300 MHz, DMSO-d₆) δ 11.75 (s, 1H), 10.04 (s, 1H), 8.39 (d, 1H),8.30 (d, 1H), 8.26 (s, 1H), 7.91 (br s, 1H), 7.69 (dd, 1H), 7.50 (d,1H), 7.43 (dd, 1H), 5.29 (s, 2H), 4.24 (m, 4H), 1.03 (m, 4H) and −0.01(m, 18H).

³¹P NMR (121 MHz, DMSO-d₆) δ 10.3.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl) phosphonooxy-4,5-dichlorobenzyl ester (29g).

Following the above procedure, 28 g (2.0 g, 3.2 mmol) gave 29 g (1.5 g,70%) as a yellow solid:

¹H NMR (300 MHz, DMSO-d₆) δ 11.85 (s, 1H), 10.08 (s, 1H), 8.42 (d, 1H),8.31 (d, 1H), 8.26 (s, 1H), 7.89 (m, 1H), 7.70 (s, 1H), 7.59 (s, 1H),7.5-7.2 (m, 2H), 5.18 (s, 2H), 4.02 (m, 4H), 0.95 (m, 4H) and 0.0 (m,18H).

¹³C NMR (75 MHz, DMSO-d₆) δ 178.6, 153.3, 148.8, 143.8, 142.4, 140.6,134.0, 130.8, 130.2 (d), 129.5, 128.3 (d), 125.8 (d), 124.4, 121.5,119.9, 64.9 (d), 60.8, 18.9 (d) and −1.5.

³¹P NMR (121 MHz, DMSO-d₆) δ 9.7.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl) phosphonooxy-5,6-dichlorobenzyl ester (29h).

Following the above procedure, 28 h (5.9 g, 9.5 mmol) gave 29 h (3.6 g,55%) as a yellow solid:

¹H NMR (300 MHz, DMSO-d₆) δ 11.82 (s, 1H), 9.97 (br s, 1H), 8.50 (m,1H), 8.41 (d, 1H), 8.27 (d, 1H), 8.23 (s, 1H), 7.72 (m, 1H), 7.67 (d,1H), 7.48 (m, 1H), 7.41 (d, 1H), 7.3−7.1 (m, 1H), 5.32 (s, 2H), 4.03 (m,4H), 0.96 (m, 4H) and −0.06 (m, 18H).

¹³C NMR (75 MHz, DMSO-d₆) δ 178.3, 153.3, 150.6 (d), 143.8, 140.8,134.0, 133.8, 133.3., 131.2, 130.3, 129.4, 126.7 (d), 124.4, 120.1,119.9 (d), 64.8 (d), 59.4, 18.9 (d) and −1.6.

³¹P NMR (121 MHz, DMSO-d₆) δ 9.6.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl) phosphonooxy-3-methylbenzyl ester (29 i).

Following the above procedure, 28 i (1.1 g, 1.9 mmol) gave 29 i (0.7 g,57%) as a yellow solid:

¹H NMR (300 MHz, DMSO-d₆) δ 11.76 (br s, 1H), 9.99 (br s, 1H), 8.42 (brs, 1H), 8.37 (m, 1H), 8.27 (br s, 1H), 8.24 (m, 1H), 8.03 (br s, 1H),7.45 (dd, 1H), 7.25 (d, 1H), 7.19 (d, 1H), 7.11 (dd, 1H), 5.30 (s, 2H),4.08 (m, 4H), 2.29 (s, 3H), 1.01 (m, 4H) and −0.03 (m, 18H).

¹³C NMR (75 MHz, DMSO-d₆) δ 178.7, 153.8, 147.5 (d), 144.4, 143.2,141.2, 134.3, 131.1, 130.7 (d), 128.9 (d), 126.4, 124.9, 124.6, 65.4(d), 62.4, 19.2 (d), 16.9 and −1.4.

³¹P NMR (121 MHz, DMSO-d₆) δ 10.4.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl) phosphonooxy-4-chlorobenzyl ester (29 j).

Following the above procedure, 28 j (10.5 g, 18.5 mmol) gave 29 j (11.8g, 97%) as a yellow solid:

¹H NMR (300 MHz, DMSO-d₆) δ 11.74 (s, 1H), 10.05 (br s, 1H), 8.4−8.3 (m,3H), 7.85 (br s, 1H), 7.56 (d, 1H), 7.5−7.4 (m, 3H), 5.21 (s, 2H), 4.22(m, 4H), 1.04 (m, 4H) and −0.01 (s, 18H).

¹³C NMR (75 MHz, DMSO-d₆) δ 153.2, 148.5, 148.4, 144.8, 144.5, 133.8,133.0, 130.8, 126.5, 126.4, 125.4, 124.1, 119.7, 67.2 (d), 60.8, 18.9(d) and −1.6.

³¹P NMR (121 MHz, DMSO-d₆) δ 9.5.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid2-bis(2-trimethylsilylethyl) phosphonooxy-4-methoxybenzyl ester (29 k).

Following the above procedure, 28 k (2.6 g, 4.5 mmol) gave 29 k (2.7 g,91%) as a yellow solid:

¹H NMR (300 MHz, DMSO-d₆) δ 11.74 (d, 1H), 9.98 (br s, 1H), 8.4−8.3 (m,3H), 7.82 (br s, 1H), 7.44 (m, 1H), 6.87 (m, 3H), 5.16 (s, 2H), 4.19 (m,4H), 3.77 (s, 3H), 1.03 (m, 4H) and −0.01 (s, 18H).

¹³C NMR (75 MHz, DMSO-d₆) δ 178.4, 160.0, 153.2, 150.2, 148.4, 144.8,144.5, 133.8, 133.0, 130.8, 124.1, 119.0, 110.4, 106.0, 66.8 (d), 61.4,55.3, 18.9 (d) and −1.6.

³¹P NMR (121 MHz, DMSO-d₆) δ 9.6.

Example 9 Preparation of Free phosphonic acids (6-17)

General Procedure. To a solution of the corresponding TMSE-protectedphosphate (24 or 29 a-k, 10 mmol) in dichloromethane (300-500 mL) wasadded trifluoroacetic acid (TFA, 20-50 mL) at 0° C. The reaction mixturewas stirred vigorously for 2 h in an ice bath. A precipitate wascollected by filtration, washed with cold dichloromethane, and thendried in vacuum. More commonly, the solvents were evaporated, and theresulting residual mixture was then dried in vacuum. The correspondingfree phosphonic acid (6-17) was obtained as a yellow solid or glassysolid.

Example 10 Preparation of disodium salt of phosphonic acid (25, 30 a-k)

General Procedure. The corresponding free phosphonic acid (6-17, 10mmol) was neutralized with an aqueous saturated sodium bicarbonate(NaHCO₃) solution (50-100 mL). The suspension was stirred for 2 h atambient temperature, and then added a minimum amount of water to makehomogenous. The aqueous solution was purified by reversed phase columnchromatography with de-ionized water. The fractions were monitored by³¹P NMR and combined. After lyophylization, the corresponding disodiumsalt (25 or 30 a-k) was obtained as a pale yellow powder.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid 2-(disodiumphosphonooxy)-5-chlorobenzyl ester (25).

Following the above procedure, 24 (1.1 g, 1.7 mmol) gave 25 (0.4 g, 49%)as a pale yellow powder:

¹H NMR (300 MHz, D₂O) δ 7.94 (br s, 2H), 7.72 (s, 1H), 7.2−7.0 (m, 3H)and 4.98 (s, 2H).

¹³C NMR (75 MHz, D₂O) δ 179.6, 157.0, 153.2, 147.2 (d), 145.2, 136.8,131.4, 130.5, 128.8, 127.8, 123.6 and 65.4.

³¹P NMR (121 MHz, D₂O) δ 14.3.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid 2-(disodiumphosphonooxy)-5-fluorobenzyl ester (30 a).

Following the above procedure, 29 a (10.5 g, 16 mmol) gave 7 (6.2 g,86%), which upon treatment with NaHCO₃ gave 30 a (4.0 g, 59%) as a paleyellow powder:

¹H NMR (300 MHz, D₂O) δ 8.2 (br s, 1H), 7.8 (br m, 1H), 7.57 (br s, 1H),7.15 (m, 1H), 6.93 (m, 1H), 6.81 (m, 1H), 6.78 (m, 1H) and 4.93 (s, 2H).

¹³C NMR (75 MHz, D₂O) δ 179.4, 161.5, 158.4, 156.5, 150.3, 147.3, 146.5,136.7, 130.8, 130.4, 127.7, 123.5, 117.5, 117.2and 65.2.

³¹P NMR (121 MHz, D₂O) δ 14.5.

¹⁹F NMR (282 MHz, D₂O) δ−57.4.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid 2-(disodiumphosphonooxy)-5-nitrobenzyl ester (30 b).

Following the above procedure, 29 b (2.1 g, 3.0 mmol) gave 30 b (1.0 g,73%) as a dark yellow powder:

¹H NMR (300 MHz, D₂O) δ 8.0−7.8 (m, 4H), 7.40 (m, 1H), 7.17 (m, 1H) and5.06 (s, 2H).

³¹P NMR (121 MHz, D₂O) δ 13.8.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid 2-(disodiumphosphonooxy)-5-methoxybenzyl ester (30 c).

Following the above procedure, 29 c (4.3 g, 16 mmol) gave 9 (2.9 g,98%), which upon treatment with NaHCO₃ gave 30 c (1.6 g, 43%) as a paleyellow powder:

¹H NMR (300 MHz, D₂O) δ 7.96 (br s, 1H), 7.70 (br s, 1H), 7.21 (br s,1H), 7.08 (br s, 1H), 6.73 (s, 2H), 5.05 (s, 2H) and 3.65 (s, 3H).

¹³C NMR (75 MHz, D₂O) δ 174.5, 151.8, 151.1, 143.4, 142.1, 141.7, 135.9,131.6, 126.4, 124.9, 122.6, 118.4, 111.7, 60.8 and 53.2.

³¹P NMR (121 MHz, D₂O) δ 14.6.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid 2-(disodiumphosphonooxy)-5-trifluoromethoxybenzyl ester (30 d).

Following the above procedure, 29 d (1.9 g, 2.6 mmol) gave 30 d (0.5 g,31%) as a pale yellow powder:

¹H NMR (300 MHz, D₂O) δ 7.93 (br s, 1H), 7.86 (br d, 1H), 7.71 (s, 1H),7.25 (d, 1H), 7.02 (m, 4H) and 5.01 (s, 2H).

¹³C NMR (75 MHz, D₂O) δ 179.5, 173.5, 157.1, 153.3, 147.1, 146.8, 145.5,141.2 (m), 136.5, 132.4 (m), 130.2 (d), 127.7 (d), 124.5, 124.0, 123.1,122.6, 121.0 and 65.4.

³¹P NMR (121 MHz, D₂O) δ 14.3.

¹⁹F NMR (282 MHz, D₂O) δ−56.3.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid 2-(disodiumphosphonooxy)-5-trifluoromethylbenzyl ester (30 e).

Following the above procedure, 29 e (3.6 g, 5.2 mmol) gave 30 e (1.3 g,45%) as a pale yellow powder:

¹H NMR (300 MHz, D₂O) δ 7.98 (br s, 1H), 7.89 (d, 1H), 7.77 (s, 1H),7.4−7.3 (m, 3H), 7.08 (m, 1H) and 5.04 (s, 2H).

³¹P NMR (121 MHz, D₂O) δ 14.0.

¹⁹F NMR (282 MHz, D₂O) δ−59.4.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid 2-(disodiumphosphonooxy)-3,5-dichlorobenzyl ester (30 f).

Following the above procedure, 29 f (4.5 g, 6.5 mmol) gave 30 f (0.8 g,24%) as a pale yellow powder:

¹H NMR (300 MHz, D₂O) δ 8.31 (br s, 1H), 7.88 (br d, 2H), 7.6−7.5 (m,2H), 7.2−6.8 (m, 5H) and 5.07 (s, 2H).

¹³C NMR (75 MHz, D₂O) δ 179.6, 156.6 (d), 149.4 (d), 147.4, 146.8 (d),136.6, 134.2, 131.5, 131.1, 130.7 (d), 130.1, 128.5, 127.7 (d) and 65.6.

³¹P NMR (121 MHz, D₂O) δ 14.4.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid 2-(disodiumphosphonooxy)-4,5-dichlorobenzyl ester (30 g).

Following the above procedure, 29 g (2.5 g, 3.0 mmol) gave 30 g (0.4 g,23%) as a pale yellow powder:

¹H NMR (300 MHz, D₂O) δ 8.07 (s, 1H), 7.99 (m, 1H), 7.85 (s, 1H), 7.39(s, 1H), 7.19 (m, 2H) and 4.99 (s, 2H).

³¹P NMR (121 MHz, D₂O) δ 14.3.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid 2-(disodiumphosphonooxy)-5,6-dichlorobenzyl ester (30 h).

Following the above procedure, 29 h (4.6 g, 6.6 mmol) gave 30 h (2.3 g,64%) as a pale yellow powder:

¹H NMR (300 MHz, D₂O) δ 8.01 (s, 1H), 7.91 (br s, 1H), 7.73 (s, 1H),7.23 (dd, 2H), 7.12 (m, 1H) and 5.18 (s, 2H).

³¹P NMR (121 MHz, D₂O) δ 14.2.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid 2-(disodiumphosphonooxy)-3-methylbenzyl ester (30 i).

Following the above procedure, 29 i (1.2 g, 1.8 mmol) gave 30 i (0.5 g,57%) as a yellow powder:

¹H NMR (300 MHz, D₂O) δ 8.11 (br s, 2H), 7.91 (m, 2H), 7.71 (m, 1H),7.00 (m, 2H), 6.84 (m, 1H), 5.22 (s, 2H) and 2.14 (s, 3H).

¹³C NMR (75 MHz, D₂O) δ 146.1, 134.6, 133.6, 131.1, 128.8, 127.9, 125.7,66.6 and 19.1.

³¹P NMR (121 MHz, D₂O) δ 14.2.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid 2-(disodiumphosphonooxy)-4-chlorobenzyl ester (30 j).

Following the above procedure, 29 j (4.2 g, 6.6 mmol) gave 30 j (1.6 g,48%) as a pale yellow powder:

¹H NMR (300 MHz, D₂O) δ 7.98 (s, 1H), 7.90 (m, 1H), 7.74 (s, 1H), 7.31(s, 1H), 7.09 (m, 3H), 6.85 (m, 1H) and 5.00 (s, 2H).

¹³C NMR (75 MHz, D₂O) δ 180.0, 157.7, 155.9, 147.6, 147.4, 142.3, 137.1,136.8, 133.2, 132.9, 128.3, 127.9, 124.9 and 65.9.

³¹P NMR (121 MHz, D₂O) δ 14.3.

(2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid 2-(disodiumphosphonooxy)-4-methoxybenzyl ester (30 k).

Following the above procedure, 29 k (2.9 g, 4.4 mmol) gave 30 k (1.2 g,54%) as a pale yellow powder:

¹H NMR (300 MHz, D₂O) δ 8.06 (s, 1H), 7.94 (s, 1H), 7.64 (s, 1H), 7.13(m, 1H), 7.0−6.8 (m, 3H), 6.43 (m, 1H), 4.06 (s, 2H) and 3.58 (s, 3H).

¹³C NMR (75 MHz, D₂O) δ 161.5, 161.3, 155.1, 133.1, 127.3, 127.0, 111.4,108.3 and 57.9.

³¹P NMR (121 MHz, D₂O) δ 14.3.

Example 11 Biological Data Antiviral Aactivity of Triapine (R¹ and R²are Both H) Plus Lamivudine Against HIV-1 Infection

MT-2 cells infected with strain IIIB of HIV (wildtype, 3TC resistant, orAZT resistant) are used for determining antiviral activities (Bridges etal., Biochem. Pharmacol. Vol. 51, 731-36, 1996). Cells are infected withvirus at a multiplicity of infection of 0.1 TCID₅₀/ml and added to wellscontaining serial 2-fold dilutions of drugs. MT-2 cells in RPMI 1640medium supplemented with 10% dialyzed fetal bovine serum and 100 μg/mLKanamycin are infected with virus and immediately added to serialdilutions of the drugs.

After 5 days, 20 μL of MTT dye (2.5 mg/mL in PBS) is added per well. Atthe end of the 4-h incubation period, 150 μL of acidified 2-propanolwith 2% NP-40 nonionic detergent is added per well. After the crystalsof dye are dissolved (usually 1-2 days), the plates are read on amicroplate reader. Using this MTT-dye reduction method (Larder et al.,(1990), Antimicrob. Agents Chemother. 34, 436-41), the percentage ofprotection can be calculated from the formula [(a-b)/(c-b)×100] in whicha is the A₅₉₅ of drug-treated virus-infected wells, b is the A₅₉₅ ofno-drug infected cells, and c is the A₅₉₅ of the no-drug uninfectedcells. The EC₅₀ was calculated from linear log 10 plots of thepercentage protection verses inhibition concentration. Concentrations ofTriapine tested are ranging from 0.1 to 0.8 micromolar whereconcentration of lamivudine are from 0.02 to 2 micromolar.

Example 12 Antiviral Activity of Triapine (R¹ and R² are Both H) PlusD4T, DDI and AZT Against HIV-1 Infection

Using the above-described experimental system, the ability of Triapineto potentiate the anti-HIV activity of the dideoxynucleoside analogs ddI(2′,3′-dideoxyinosine), D4T (2′,3′-dideoxydidehydrothymidine) and AZT(3′-azidothymidine) was examined in HIV-infected MT-2 cells (asdescribed above). Isobolograms, describing the interactions, wereplotted according to the method described in Bridges et al., Biochem.Pharmacol., 51, 731-736 (1996). The results, presented in FIG. 4,evidence that triapine elicits synergistic anti-HIV activites whencombined with D4T and ddI and interacts in an additive fashion whencombined with AZT.

Example 13 Antiviral Activity of Triapine Plus Lamivudine Against HBVInfection

The effects of drugs on HBV viral DNA replication are assessed asdescribed by Doong et al. (Proc. Natl. Acad. Sci. USA 88, 8495-99,1991). A human hepatoma cell line carrying HBV (designated 2.2.15) isused in this study (Price et al., (1989) Proc. Natl. Acad. Sci. USA 86,8541-44). Six-day-old cultures are treated with various concentrationsof the drug in culture medium (minimal essential medium with Earle'ssalts and 10% fetal bovine serum [MEME]). The drugs are left in theculture medium for 3 days, and then the medium is aspirated and freshmedium containing the same concentration of the drugs are added. At theend of the subsequent 3-day period, the culture medium is harvested. Analiquot of the culture medium (5 μl) is used for the estimation of theHBV surface antigen (HBVsAg) (as described below). The culture medium isprocessed to obtain virions by polyethylene glycol precipitation. Theviral DNA recovered from the secreted particles is subjected to Southernblot analysis (Sambrook et al., (1989) Molecular cloning laboratorymanual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). Inhibition of viral DNA replication is determined by comparisonof the viral DNAs from drug-treated and nontreated cultures. Southernblot analysis of the DNA is performed, and the level of inhibition isdetermined by hybridization of the blots to an HBV-specific probefollowed by autoradiography. Quantitation of the autoradiographs isperformed by densitometric scans on a densitometer. The percentage ofinhibition can be calculated from the formula [1-(a-b)/(c-b)×100] inwhich a is the densitometric unit of drug-treated virus-infected wells,b is the densitometric unit of no-drug infected cells, and c is thedensitometric unit of the no-drug uninfected cells. The ED₅₀ wascalculated from linear log 10 plots of percentage inhibition verses theinhibition concentration.

Example 14 Antiviral Activity of Triapine Plus Acyclovir Against HSVInfection

To evaluate the antiviral activities of Triapine, acyclovir, andcombination thereof, HeLa S₃ cells are seeded in 25-cm² flasks and areused as host cells for virus infection (laboratory strains of virusHSV-1, HSV-2, wild type (Darby et al., (1982) Nature, 289, 81-3; Cheng,et al., Antimicrob. Agents Chemother. 18, 957-61, 1980). After a 1-hradsorption period with virus at 5-10 plaque-forming units per cell, themonolayers are rinsed with phosphate-buffered saline, followed by theaddition of 5 ml of growth medium containing various concentrations ofdrug. Concentrations of Triapine tested range from 0.1 to 1.0 micromolarwhere concentrations of acyclovir are from 1-50 micromolar. The cellsare incubated at 37° C. for 48 hr and then are stored frozen at −70° C.until titration. The amounts of HSV contained in cells are determinedwith Vero cells using the methods described by Cheng et al. (Cheng, etal., Antimicrob. Agents Chemother. 18, 957-61, 1980).

The results of the above-test are presented in the following Table 1,below, which presents the antiviral activity of Triapine, at low,non-toxic clinically achievable concentrations of 0.3, 0.6 and 1.0 μM,in combination with acyclovir against HSV-1 (KOS) and HSV-2 (333)strains of herpes simplex virus in a virus yield assay. The experimentalresults evidence that in the presence of as little as 0.3 and 0.6 μMTriapine, the EC₉₀ of ACV decreased by 10- to greater than 24-fold forHSV-1. A greater than 4-fold decrease in the EC₉₀ of ACV against HSV-2is observed with 0.6 μM Triapine. These data evidence that Triapine maymarkedly potentiate the anti-HSV activity of anti-HSV nucleoside-typeanalogs such as ACV in treating HSV infections. TABLE 1 Virus YieldAssay in HSV-Infected Vero Cells EC₉₀ (μM) DRUG HSV-1 HSV-2 ACV 29.336.5 Triapine >1 >1 0.3 ACV + 0.3 μM Triapine 2.9 4.7 0.6 ACV + 0.6 μMTriapine 1.2 4.8    ACV + 1.0 μM Triapine <0.005 2.2

It is to be understood by those skilled in the art that the foregoingdescription and examples are illustrative of practicing the presentinvention, but are in no way limiting. Variations of the detailpresented herein may be made without departing from the spirit and scopeof the present invention as defined by the following claims.

1. A method of treating a viral infection in a patient comprisingadministering to said patient a pharmaceutical composition comprising ananti-viral effective amount of at least one compound according to thestructure:

Where R¹ is H or a C₁-C₃ alkyl group, preferably H or CH₃; R² is H orCO₂R³; R³ is CHRR′ or

where R is H, CH₃, CH₂CH₃, CH₂CH₂CH₃, i-propyl; R′ is a free acidphosphate, phosphate salt or S—S—R″ group; R″ is CH₂CH₂NHR⁴, CH₂CH₂OH,CH₂COOR⁵, ortho- or para-substituted C₁-C₃ alkylphenyl or ortho or paranitrophenyl; R⁴ is H or a C₁-C₁₈ acyl group (preferably, a C₁-C₄ group),benzoyl, or a substituted benzoyl group; R⁵ is H, a C₁-C₁₈ alkyl group(preferably, a C₁-C₃ group), phenyl, substituted phenyl, benzyl, or asubstituted benzyl group; R⁶, R⁷, R⁸, R⁹ and R¹⁰ are independentlyselected from H, a free acid phosphate, a phosphate salt, or an S—S—R″group, a C₁-C₃ alkyl group, F, Cl, Br, I, OCH₃, OCF₃, CF₃, NO₂, CN,SO₂CF₃, SO₂CH₃, COOCH₃, SF₅, COCH₃, NH₂, N(CH₃)₂, SCH₃ or OH; With theproviso that when any two of R⁶, R⁷, R⁸, R⁹ and R¹⁰ are other than H,the other of R⁶, R⁷, R⁸, R⁹ and R¹⁰ are H, and with the proviso that nomore than one of R⁶, R⁷, R⁸, R⁹ and R¹⁰ is a free acid phosphate, aphosphate salt or S—S—R″. 2-4. (canceled)
 5. The method according toclaim 1 wherein R¹ and R² are H.
 6. The method according to claim 1wherein said viral infection is caused by an agent selected from thegroup consisting of human immunodeficiency viruses 1 and 2 (HIV-1 andHIV-2), human T-cell leukemia viruses 1 and 2 (HTLV-1 and HTLV-2),respiratory syncytial virus (RSV), human papilloma virus (HPV),adenovirus, hepatitis B virus (HBV), hepatitis C virus (HCV),Epstein-Barr virus (EBV), varicella zoster virus (VZV), cytomegalovirus(CMV), herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), human herpesvirus 8 (HHV-8, also known as Kaposi's sarcoma-associated virus) andflaviviruses, including Yellow Fever virus, Dengue virus, JapaneseEncephalitis and West Nile viruses.
 7. A method of treating a viralinfection comprising administering in combination, at least one compoundaccording to the structure:

Where R¹ is H or a C₁-C₃ alkyl group, preferably H or CH₃; R² is H orCO₂R³; R³ is CHRR′ or

where R is H, CH₃, CH₂CH₃, CH₂CH₂CH₃, i-propyl; R′ is a free acidphosphate, phosphate salt or S—S—R″ group; R″ is CH₂CH₂NHR⁴, CH₂CH₂OH,CH₂COOR⁵, ortho- or para-substituted C₁-C₃ alkylphenyl or ortho or paranitrophenyl; R⁴ is H or a C₁-C₁₈ acyl group (preferably, a C₁-C₄ group),benzoyl, or a substituted benzoyl group; R⁵ is H, a C₁-C₁₈ alkyl group(preferably, a C₁-C₃ group), phenyl, substituted phenyl, benzyl, or asubstituted benzyl group; R⁶, R⁷, R⁸, R⁹ and R¹⁰ are independentlyselected from H, a free acid phosphate, a phosphate salt, or an S—S—R″group, a C₁-C₃ alkyl group, F, Cl, Br, I, OCH₃, OCF₃, CF₃, NO₂, CN,SO₂CF₃, SO₂CH₃, COOCH₃, SF₅, COCH₃, NH₂, N(CH₃)₂, SCH₃ or OH; With theproviso that when any two of R⁶, R⁷, R⁸, R⁹ and R¹⁰ are other than H,the other of R⁶, R⁷, R⁸, R⁹ and R¹⁰ are H, and with the proviso that nomore than one of R⁶, R⁷, R⁸, R⁹ and R¹⁰ is a free acid phosphate, aphosphate salt or S—S—R″ and at least one anti-viral agent whichinhibits the growth or replication of viruses by a mechanism other thanby inhibition of viral ribonucleotide reductase. 8-10. (canceled) 11.The method according to claim 7 wherein R¹ and R² are H.
 12. The methodaccording to claim 7 wherein said viral infection is caused by an agentselected from the group consisting of human immunodeficiency viruses 1and 2 (HIV-1 and HIV-2), human T-cell leukemia viruses 1 and 2 (HTLV-1and HTLV-2), respiratory syncytial virus (RSV), human papilloma virus(HPV), adenovirus, hepatitis B virus (HBV), hepatitis C virus (HCV),Epstein-Barr virus (EBV), varicella zoster virus (VZV), cytomegalovirus(CMV), herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), human herpesvirus 8 (HHV-8, also known as Kaposi's sarcoma-associated virus) andflaviviruses, including Yellow Fever virus, Dengue virus, JapaneseEncephalitis and West Nile viruses.
 13. The method according to claim 7wherein said anti-viral agent is selected from the group consisting ofacyclic nucleosides, interferons, reverse transcriptase inhibitors,nucleoside transport inhibitors 2′,3′-dideoxynucleosides, 3TC, AZT,2′,3′-dideoxycytidine, 2′,3′-dideoxyadenosine, 2′,3′-dideoxyinosine,2′,3′-dideoxythymidine, 2′,3′-dideoxy-2′,3′-didehydrothymidine and2′,3′-dideoxy-2′,3′-didehydrocytidine, β-LFddC, β-LFd4C, β-Ld4C,tenofovir DF, adefovir, dipivoxil, immunomodulators such as interleukinII (IL2) and granulocyte macrophage colony stimulating factor (GM-CSF),erythropoetin, ampligen, thymodulin, thymopentin, foscarnet, ribavirinand inhibitors of HIV binding to CD4 receptors such as soluble CD4, CD4fragments, CD4 hybrid molecules and glycosylation inhibitors such as2-deoxy-D-glucose, castanospermine and 1-deoxynojirimycin. 14.(canceled)
 15. A pharmaceutical composition comprising an anti-viraleffective amount of at least one compound according to the structure:

Where R¹ is H or a C₁-C₃ alkyl group, preferably H or CH₃; R² is H orCO₂R³; R³ is CHRR′ or

where R is H, CH₃, CH₂CH₃, CH₂CH₂CH₃, i-propyl; R′ is a free acidphosphate, phosphate salt or S—S—R″ group; R″ is CH₂CH₂NHR⁴, CH₂CH₂OH,CH₂COOR⁵, ortho- or para-substituted C₁-C₃ alkylphenyl or ortho or paranitrophenyl; R⁴ is H or a C₁-C₁₈ acyl group (preferably, a C₁-C₄ group),benzoyl, or a substituted benzoyl group; R⁵ is H, a C₁-C₁₈ alkyl group(preferably, a C₁-C₃ group), phenyl, substituted phenyl, benzyl, or asubstituted benzyl group; R⁶, R⁷, R⁸, R⁹ and R¹⁰ are independentlyselected from H, a free acid phosphate, a phosphate salt, or an S—S—R″group, a C₁-C₃ alkyl group, F, Cl, Br, I, OCH₃, OCF₃, CF₃, NO₂, CN,SO₂CF₃, SO₂CH₃, COOCH₃, SF₅, COCH₃, NH₂, N(CH₃)₂, SCH₃ or OH; With theproviso that when any two of R⁶, R⁷, R⁸, R⁹ and R¹⁰ are other than H,the other of R⁶, R⁷, R⁸, R⁹ and R¹⁰ are H, and with the proviso that nomore than one of R⁶, R⁷, R⁸, R⁹ and R¹⁰ is a free acid phosphate, aphosphate salt or S—S—R″ and at least one other anti-viral agent whichinhibits the growth or replication of viruses by a mechanism other thanby inhibition of viral ribonucleotide reductase. 16-18. (canceled) 19.The composition according to claim 15 wherein said composition is usedto treat a viral infection caused by an agent selected from the groupconsisting of human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2),human T-cell leukemia viruses 1 and 2 (HTLV-1 and HTLV-2), respiratorysyncytial virus (RSV), human papilloma virus (HPV), adenovirus,hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus(EBV), varicella zoster virus (VZV), cytomegalovirus (CMV), herpessimplex virus 1 and 2 (HSV-1 and HSV-2), human herpes virus 8 (HHV-8,also known as Kaposi's sarcoma-associated virus) and flaviviruses,including Yellow Fever virus, Dengue virus, Japanese Encephalitis andWest Nile viruses.
 20. The composition according to claim 15 whereinsaid other anti-viral agent is selected from the group consisting ofacyclic nucleosides such as acyclovir or ganciclovir, interferons suchas alpha, beta or gamma-interferon, reverse transcriptase inhibitors andnucleoside transport inhibitors such as dipyridamole,2′,3′-dideoxynucleosides, 3TC, AZT, 2′,3′-dideoxycytidine,2′,3′-dideoxyadenosine, 2′,3′-dideoxyinosine, 2′,3′-dideoxythymidine,2′,3′-dideoxy-2′,3′-didehydrothymidine and2′,3′-dideoxy-2′,3′-didehydrocytidine, β-LFddC, β-LFd4C, β-Ld4C,tenofovir DF, adefovir, dipivoxil, immunomodulators such as interleukinII (IL2) and granulocyte macrophage colony stimulating factor (GM-CSF),erythropoetin, ampligen, thymodulin, thymopentin, foscarnet, ribavirinand inhibitors of HIV binding to CD4 receptors e.g. soluble CD4, CD4fragments, CD4 hybrid molecules, glycosylation inhibitors such as2-deoxy-D-glucose, castanospermine and 1-deoxynojirimycin.
 21. Thecomposition according to claim 15 wherein R¹ and R² are H.
 22. Thecomposition according to claim 21 wherein said other anti-viral agent isselected from the group consisting of 3TC, DDI, D4T, P-LFd4C, β-LFddC,AZT, acyclovir and gancyclovir.
 23. The composition according to claim21 wherein said other anti-viral agent is selected from the groupconsisting of 3TC, DDI, D4T and AZT. 24-35. (canceled)
 36. A method oftreating an HIV infection in a patient in need thereof comprisingadministering to said patient in combination, an effective amount of atleast one compound according to the structure:

Where R¹ and R² are both H, with an effective amount of at least oneanti-HIV agent selected from the group consisting of ddI and D4T, saidadministration producing a synergistic effect in treating said patient.37. The method according to claim 36 wherein said anti-HIV agent is ddI.38. The method according to claim 36 wherein said anti-HIV agent is D4T.39. A method of treating an HSV infection in a patient in need thereofcomprising administering in combination, an effective amount of at leastone compound according to the structure:

Where R¹ and R² are both H, with an effective amount of acyclovir.